Information

What is 'calcium conductance'?


What is the meaning of calcium conductance in ion channels. I encountered this in the following text:

It was established that the µ and δ opioid receptors open potassium channels, which results in reduction of calcium conductance (Simon, 2005).

Also why should opening a potassium channel reduce calcium conductance. How are they related ?

Reference:

Simon, E. J. (2005). Opiates: Neurobiology. In J. H. Lowinson, P. Ruiz, R. B. Millman & J. G. Langrod (Eds.), Substance abuse : a comprehensive textbook (4th ed., pp. xxiv, 1421 p.). Philadelphia: Lippincott Williams & Wilkins.


Conductance is the inverse of resistance, and measures how much of a given substance flows throught a channel. In this context, it means how many calcium ions enter the cell in a period of time.

There are at least two ways potassium channels may prevent the calcium to enter in the cell.

1) Potassium intake by ion channels decrease the membrane potential, restoring it to its rest state. Since many calcium channels are voltage-dependent, a reduction of the membrane potential would close them, effectively decreasing the calcium conductance.

2) Potassium channels may be coupled to different signaling pathways (i.e. G proteins), wich may affect indirectly other calcium channels.

In the case of opioid receptors it seems to be the first mechanism. With potassium channels open, the neuron is less likely to be activated, because it needs higher stimulation to reach the action potential. Calcium channels open in neurons mainly during action potential events (althought they may exist other calcium channels that open in other conditions, i. e in response to hormones or neuromodulators).


Neuronal calcium signaling

In neurons calcium plays a dual role as a charge carrier and an intracellular messenger. Calcium signals regulate various developmental processes and have a key role in apoptosis, neurotransmitter release and membrane excitability. How can one ubiquitous intracellular messenger regulate so many different vital processes in parallel, but also work independently? The answer lies in the versatility of the calcium signaling mechanisms in terms of amplitude and spatiotemporal patterning within a neuron. Here we describe some of the main contributors to neuronal calcium signaling.

Voltage-gated calcium channels (VGCCs)

Voltage-gated calcium channels are the primary mediators of depolarization-induced calcium entry into neurons. There is great diversity in calcium channel subtypes due to multiple genes that encode calcium channel subunits, alternative splicing and coassembly with a variety of ancillary calcium channel subunits. This allows VGCCs to carry out distinct roles in specific neuronal subtypes and at particular subcellular loci.

Under resting conditions, intracellular calcium concentrations lie in the 100 nM range due to calcium-buffering molecules and sequestration into intracellular calcium stores. VGCCs opening result in calcium influx along the electrochemical gradient, leading to a transient, localized elevation of intracellular calcium concentration into the high micromolar range. This in turn triggers a wide range of calcium-dependent processes that include gene transcription, neurotransmitter release, neurite outgrowth, and the activation of calcium-dependent enzymes such as calmodulin-dependent protein kinase II and protein kinase C.

Calcium release from internal stores

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) through calcium release channels inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). Calcium signals resulting from calcium release from internal stores have been found in various types of neurons at different developmental stages. While IP3-mediated calcium release is mostly triggered by neurotransmitters such as glutamate (see below), RyRs can be activated by elevations of the cytosolic calcium concentration. This calcium-induced calcium release mediated by RyR can contribute to the amplification of the calcium influx generated by action potential firing in neurons. Both IP 3Rs and RyRs are regulated by calcium itself along with other intracellular factors. This dependence on calcium establishes a feedback loop coordinating calcium influx from the internal stores into the cytosol. In the case of IP3Rs, calcium influx plays an essential role for in generating calcium waves in neocortical and other types of neurons.

NMDA receptors

NMDA receptors are ionotropic glutamate receptors and mediate a major part of the postsynaptic calcium influx in the dendritic spines of various neuronal cell types and cortex. This rise in spinal calcium concentration is particularly important for the long-term modification of synaptic strength. NMDA receptor channels are nonspecific cation channels that are permeable for sodium, potassium, and calcium ions.

Calcium-permeable AMPA receptors

Calcium-permeable AMPA receptors are another class of ionotropic glutamate receptors. They are found in many forms of aspiny GABAergic neurons and are characterized by the lack of a GluR2 receptor subunit. GluR2-lacking AMPA receptors are permeable for sodium, calcium, potassium and zinc ions. Calcium-permeable AMPA receptors have a high conductance in response to tetanic stimulation and enables individual neurons to produce different types of responses to distinct synaptic inputs. Importantly, the presence of GluR2-containing (native AMPA receptors) and GluR2-lacking AMPA receptors (calcium-permeable AMPA receptors) is not static, but is highly regulated, particularly in response to neuronal activity. Thus, permeability of AMPA receptors to calcium is dynamic within a given neuron and can therefore contribute to synaptic plasticity mechanisms in aspiny neurons.

Direct calcium entry through AMPA receptors is capable of triggering neuronal death. Therefore the divergence in relative calcium permeability of AMPA receptors between different neuronal cell types could be an important determinant of selective neuronal vulnerability.

Metabotrophic glutamate receptors (mGluRs)

mGluRs are 7-transmembrane G protein-coupled receptors that are broadly distributed within the central and peripheral nervous sytems. They are classified in group I, II, and III mGluRs, are expressed in a cell-type-specific fashion, and exert diverse physiological roles. The receptor classes differ in their downstream signaling mechanisms for example, mGluR1 are coupled to the Gq protein. In expression systems, the mGluR1 subtype of this group mediates both an increase in intracellular calcium as well as a TRPC3-dependent inward current. Upon activation of mGluR1, phospholipase C mediates the generation of IP3, which binds to receptors in the ER and induces calcium release. In contrast, an activation of native mGluR5 in neurons induces different cellular effects. In hippocampal neurons, mGluR5 elicits a single peaked intracellular calcium response, whereas in the neocortex it induces intracellular calcium oscillations.

Summary

The major challenge in the analysis of the various sources of neuronal calcium signaling is that they are generally not active one at a time, but have overlapping activities with strong interactions. Therefore, calcium imaging is invaluable for decoding the specific signaling mechanisms in neurons.


Contents

Structurally, BK channels are homologous to voltage- and ligand-gated potassium channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain for the binding of intracellular calcium and magnesium. [5] Each monomer of the channel-forming alpha subunit is the product of the KCNMA1 gene (also known as Slo1). The Slo1 subunit has three main structural domains, each with a distinct function: the voltage sensing domain (VSD) senses membrane potential across the membrane, the cytosolic domain (senses calcium concentration, Ca²⁺ ions), and the pore-gate domain (PGD) which opens and closes to regulate potassium permeation. The activation gate resides in the PGD, which is located at either the cytosolic side of S6 or the selectivity filter (selectivity is the preference of a channel to conduct a specific ion). [5] The Voltage sensing domain and pore-gated domain are collectively referred as the membrane-spanning domains and are formed by transmembrane segments S1-S4 and S5-S6, respectively. Within the S4 helix contains a series of positively charged residues which serve as the primary voltage sensor. [6]

BK channels are quite similar to voltage gated K⁺ channels, however, in BK channels only one positively charged residue (Arg213) is involved in voltage sensing across the membrane. [5] Also unique to BK channels is an additional S0 segment, this segment is required for β subunit modulation. [7] [8] and voltage sensitivity. [9]

The Cytosolic domain is composed of two RCK (regulator of potassium conductance) domains, RCK1 and RCK2. These domains contain two high affinity Ca²⁺ binding sites: one in the RCK1 domain and the other in a region termed the Ca²⁺ bowl that consists of a series of Aspartic acid (Asp) residues that are located in the RCK2 domain. The Mg²⁺ binding site is located between the VSD and the cytosolic domain, which is formed by: Asp residues within the S0-S1 loop, Asparagine residues in the cytosolic end of S2, and Glutamine residues in RCK1. [5] In forming the Mg²⁺ binding site, two residues come from the RCK1 of one Slo1 subunit and the other two residues come from the VSD of the neighboring subunit. In order for these residues to coordinate the Mg²⁺ ion, the VSD and cytosolic domain from neighboring subunits must be in close proximity. [5] Modulatory beta subunits (encoded by KCNMB1, KCNMB2, KCNMB3, or KCNMB4) can associate with the tetrameric channel. There are four types of β subunits (β1-4), each of which have different expression patterns that modify the gating properties of the BK channel. The β1 subunit is primarily responsible for smooth muscle cell expression, both β2 and β3 subunits are neuronally expressed, while β4 is expressed within the brain. [5] The VSD associates with the PGD via three major interactions:

  1. Physical connection between the VSD and PGD through the S4-S5 linker.
  2. Interactions between the S4-S5 linker and the cytosolic side of S6.
  3. Interactions between S4 and S5 of a neighboring subunit.

BK channels are associated and modulated by a wide variety of intra- and extracellular factors, such as auxiliary subunits (β, γ), Slobs (slo binding protein), phosphorylation, membrane voltage, chemical ligands (Ca²⁺, Mg²⁺), PKC, The BK α-subunits assemble 1:1 with four different auxiliary types of β-subunits (β1, β2, β3 or β4). [10]

Trafficking to and expression of BK channels in the plasma membrane has been found to be regulated by distinct splicing motifs located within the intracellular C-terminal RCK domains. In particular a splice variant that excluded these motifs prevented cell surface expression of BK channels and suggests that such a mechanism impacts physiology and pathophysiology. [10]

BK channels in the vascular system are modulated by agents naturally produced in the body, such as angiotensin II (Ang II), high glucose or arachidonic acid (AA) which is modulated in diabetes by oxidative stress (ROS). [10]

A weaker voltage sensitivity allows BK channels to function in a wide range of membrane potentials. This ensures that the channel can properly perform its physiological function. [11]

Inhibition of BK channel activity by phosphorylation of S695 by protein kinase C (PKC) is dependent on the phosphorylation of S1151 in C terminus of channel alpha-subunit. Only one of these phosphorylations in the tetrameric structure needs to occur for inhibition to be successful. Protein phosphatase 1 counteracts phosphorylation of S695. PKC decreases channel opening probability by shortening the channel open time and prolonging the closed state of the channel. PKC does not affect the single-channel conductance, voltage dependence, or the calcium sensitivity of BK channels. [11]

BK channels are synergistically activated through the binding of calcium and magnesium ions, but can also be activated via voltage dependence. [10] Ca²⁺ - dependent activation occurs when intracellular Ca²⁺ binds to two high affinity binding sites: one located in the C-terminus of the RCK2 domain (Ca²⁺ bowl), and the other located in the RCK1 domain. [5] The binding site within the RCK1 domain has somewhat of a lower affinity for calcium than the Ca²⁺ bowl, but is responsible for a larger portion of the Ca²⁺ sensitivity. [12] Voltage and calcium activate BK channels using two parallel mechanisms, with the voltage sensors and the Ca²⁺ bindings sites coupling to the activation gate independently, except for a weak interaction between the two mechanisms. The Ca²⁺ bowl accelerates activation kinetics at low Ca²⁺ concentrations while RCK1 site influences both activation and deactivation kinetics. [11] One mechanism model was originally proposed by Monod, Wyman, and Changeux, known as the MWC model. The MWC model for BK channels explains that a conformational change of the activation gate in channel opening is accompanied by a conformational change to the Ca²⁺ binding site, which increases the affinity of Ca²⁺ binding. [12]

Magnesium-dependent activation of BK channels activates via a low-affinity metal binding site that is independent from Ca²⁺-dependent activation. The Mg²⁺ sensor activates BK channels by shifting the activation voltage to a more negative range. Mg²⁺ activates the channel only when the voltage sensor domain stays in the activated state. The cytosolic tail domain (CTD) is a chemical sensor that has multiple binding sites for different ligands. The CTD activates the BK channel when bound with intracellular Mg²⁺ to allow for interaction with the voltage sensor domain (VSD). [11] Magnesium is predominantly coordinated by six oxygen atoms from the side chains of oxygen-containing residues, main chain carbonyl groups in proteins, or water molecules. [12] D99 at the C-terminus of the S0-S1 loop and N172 in the S2-S3 loop contain side chain oxygens in the voltage sensor domain that are essential for Mg²⁺ binding. Much like the Ca²⁺-dependent activation model, Mg²⁺-dependent activation can also be described by an allosteric MCW gating model. While calcium activates the channel largely independent of the voltage sensor, magnesium activates the channel by channel by an electrostatic interaction with the voltage sensor. [12] This is also known as the Nudging model, in which Magnesium activates the channel by pushing the voltage sensor via electrostatic interactions and involves the interactions among side chains in different structural domains. [5] Energy provided by voltage, Ca²⁺, and Mg²⁺ binding will propagate to the activation gate of BK channels to initiate ion conduction through the pore. [5]

Cellular level Edit

BK channels help regulate both the firing of neurons and neurotransmitter release. [13] This modulation of synaptic transmission and electrical discharge at the cellular level is due to BK channel expression in conjunction with other potassium-calcium channels. [10] The opening of these channels causes a drive towards the potassium equilibrium potential and thus play a role in speeding up the repolarization of action potentials. [10] This would effectively allow for more rapid stimulation. [10] There is also a role played in shaping the general repolarization of cells, and thus after hyperpolarization (AHP) of action potentials. [14] The role that BK channels have in the fast phase of AHP has been studied extensively in the hippocampus. [14] It can also play a role in inhibiting the release of neurotransmitters. [15] There are many BK channels in Purkinje cells in the cerebellum, thus highlighting their role in motor coordination and function. [14] Furthermore, BK channels play a role in modulating the activity of dendrites as well as astrocytes and microglia. [15] They not only play a role in the CNS (central nervous system) but also in smooth muscle contractions, the secretion of endocrine cells, and the proliferation of cells. [13] Various γ subunits during early brain development are involved in neuronal excitability and in non-excitable cells they often are responsible as a driving force of calcium. [10] Therefore, these subunits can be targets for therapeutic treatments as BK channel activators. [10] There is further evidence that inhibiting BK channels would prevent the efflux of potassium and thus reduce the usage of ATP, in effect allowing for neuronal survival in low oxygen environments. [10] BK channels can also function as a neuronal protectant in terms such as limiting calcium entry into the cells through methionine oxidation. [10]

Organ level Edit

BK channels also play a role in hearing. [14] This was found when the BK ɑ-subunit was knocked out in mice and progressive loss of cochlear hair cells, and thus hearing loss, was observed. [14] BK channels are not only involved in hearing, but also circadian rhythms. Slo binding proteins (Slobs) can modulate BK channels as a function of circadian rhythms in neurons. [10] BK channels are expressed in the suprachiasmatic nucleus (SCN), which is characterized to influence the pathophysiology of sleep. [14] BK channel openers can also have a protective effect on the cardiovascular system. [10] At a low concentration of calcium BK channels have a greater impact on vascular tone. [10] Furthermore, the signaling system of BK channels in the cardiovascular system have an influence on the functioning of coronary blood flow. [10] One of the functions of the β subunit in the brain includes inhibition of the BK channels, allowing for the slowing of channel properties as well as the ability to aid in prevention of seizures in the temporal lobe. [10]

Bodily function level Edit

Mutations of BK channels, resulting in a lower amount of expression in mRNA, is more common in people who are mentally challenged (via hypofunction [15] ), schizophrenic or autistic. [10] Moreover, increased repolarization caused by BK channel mutations may lead to dependency of alcohol initiation of dyskinesias, epilepsy or paroxysmal movement disorders. [10] Not only are BK channels important in many cellular processes in the adult it also is crucial for proper nutrition supply to a developing fetus. [10] Thus, estrogen can cause an increase in the density of BK channels in the uterus. [10] However, increased expression of BK channels have been found in tumor cells, and this could influence future cancer therapy, discussed more in the pharmacology section. [10] BK channels are ubiquitous throughout the body and thus have a large and vast impact on the body as a whole and at a more cellular level, as discussed.

Potential issues Edit

Several issues arise when there is a deficit in BK channels. Consequences of the malfunctioning BK channel can affect the functioning of a person in many ways, some more life threatening than others. BK channels can be activated by exogenous pollutants and endogenous gasotransmitters carbon monoxide, [16] [17] nitric oxide, and hydrogen sulphide. [18] Mutations in the proteins involved with BK channels or genes encoding BK channels are involved in many diseases. A malfunction of BK channels can proliferate in many disorders such as: epilepsy, cancer, diabetes, asthma, and hypertension. [13] Specifically, β1 defect can increase blood pressure and hydrosaline retention in the kidney. [13] Both loss of function and gain of function mutations have been found to be involved in disorders such as epilepsy and chronic pain. [15] Furthermore, increases in BK channel activation, through gain-of-function mutants and amplification, has links to epilepsy and cancer. [13] Moreover, BK channels play a role in tumors as well as cancers. In certain cancers gBK, a variant ion channel called glioma BK channel, can be found. [14] It is known that BK channels do in some way influence the division of cells during replication, which when unregulated can lead to cancers and tumors. [14] Moreover, an aspect studied includes the migration of cancer cells and the role in which BK channels can facilitate this migration, though much is still unknown. [14] Another reason why BK channel understanding is important involves its role in organ transplant surgery. This is due to the activation of BK channels influencing repolarization of the resting membrane potential. [10] Thus, understanding is crucial for safety in effective transplantation.

Current developments Edit

BK channels can be used as pharmacological targets for the treatment of several medical disorders including stroke [19] and overactive bladder. [20] There have been attempts to develop synthetic molecules targeting BK channels, [21] however their efforts have proven largely ineffective thus far. For instance, BMS-204352, a molecule developed by Bristol-Myers Squibb, failed to improve clinical outcome in stroke patients compared to placebo. [22] However, there have been some success from the agonist to BKCa channels, BMS-204352, in treating deficits observed in Fmr1 knockout mice, a model of Fragile X syndrome. [23] [24] BK channels also function as a blocker in ischemia and are a focus in investigating its use as a therapy for stroke. [10]

Future directions Edit

There are many applications for therapeutic strategies involving BK channels. There has been research displaying that a blockage of BK channels results in an increase in neurotransmitter release, effectively indicating future therapeutic possibilities in cognition enhancement, improved memory, and relieving depression. [13] A behavioral response to alcohol is also modulated by BK channels, [10] therefore further understanding of this relationship can aid treatment in patients who are alcoholics. Oxidative stress on BK channels can lead to the negative impairments of lowering blood pressure through cardiovascular relaxation have on both aging and disease. [10] Thus, the signaling system can be involved in treating hypertension and atherosclerosis [10] through targeting of the ɑ subunit to prevent these detrimental effects. Furthermore, the known role that BK channels can play in cancer and tumors is limited. Thus, there is not a lot of current knowledge regarding specific aspects of BK channels that can influence tumors and cancers. [14] Further study is crucial, as this could lead to immense development in treatments for those suffering from cancer and tumors. It is known that epilepsies are due to over-excitability of neurons, which BK channels have a large impact on controlling hyperexcitability. [4] Therefore, understanding could influence the treatment of epilepsy. Overall, BK channels are a target for future pharmacological agents that can be used for benevolent treatments of disease.


Membrane Potential

Introduction

An excitable membrane has a stable potential when there is no net ion current flowing across the membrane. Two factors determine the net flow of ions across an open ionic channel: the membrane potential and the differences in ion concentrations between the intracellular and the extracellular spaces. Because cells have negative intracellular potentials, the electrical force will tend to direct positively charged ions (cations such as sodium, potassium, and calcium) to flow into a cell. Hence, electrical forces will direct an inward flow of sodium, potassium, and calcium ions and an outward flow of chloride ions. The direction of ion movement produced by the ‘concentration force’ depends on the concentration differences for the ion between the intracellular and the extracellular compartments. Sodium, calcium, and chloride ions have higher extracellular concentrations compared with intracellular concentrations. The intracellular concentration of potassium is greater than the extracellular concentration. Concentration forces direct an inward flow of sodium, calcium, and chloride ions and an outward flow of potassium ions. The membrane potential at which the electrical and concentration forces are balanced for a given ion is called the equilibrium or Nernst potential for a given ion. At the equilibrium potential, inward and outward current movements are balanced for a specific ion due to balancing of the electrical and concentration forces. For a given cation, at membrane potentials that are negative compared with the equilibrium potential, ions flow into the cell, and at membrane potentials that are more positive than the equilibrium potential, current carried by the specific ion will flow out of the cell. The direction of current movement for a specific ion always tends to bring the membrane potential back to the equilibrium potential for that specific ion. Examples of approximate equilibrium potentials for ions in skeletal muscle are shown in Table 1 .

Table 1 . Equilibrium potentials

IonEquilibrium potential (mV)
Sodium65
Potassium−105
Calcium&gt100
Chloride−95 (Resting potential)
Resting potential−95

The membrane potential represents a balance among the equilibrium potentials of the ions to which the membrane is permeable. The greater the conductance of an ion, the more that ion will influence the membrane potential of the cell. The principal conductances responsible for establishing the resting membrane potential are that of chloride, potassium, and sodium. Chloride conductance is large in skeletal muscle fibers, in which it is mediated by skeletal muscle chloride channels. Peripheral nerve fibers have smaller chloride conductances. In skeletal muscle, chloride is the dominant membrane conductance, accounting for approximately 80% of the resting membrane conductance. Chloride channels in skeletal muscle are unusual in that they are gated by the presence of ions at the intracellular and extracellular orifices rather than by the membrane potential. The channel is likely to open when a chloride ion presents itself. The unique gating properties of chloride channels result in the chloride ions being distributed across the membrane in accord with the membrane potential. Consequently, chloride conductance does not set the membrane potential.

Instead, chloride conductance acts as a brake to make it more difficult for the membrane to depolarize. Therefore, chloride conductance provides an important stabilizing influence on the membrane potential.

The dominant ion in setting the resting membrane potential is potassium. Potassium conductance accounts for approximately 20% of the resting membrane conductance in skeletal muscle and accounts for most of the resting conductance in neurons and nerve fibers. This is primarily attributable to nongated ion channels, which are made up of inward rectifier and ‘slow-leak’ channels. Inward rectifier channels are responsible for maintaining the membrane potential in the absence of an excitation electrical current. It is the nongated ion channels that are responsible for differences in the electrical response of various cell types. For example, neurons, which contain nongated ion channels for potassium, sodium, and chloride, have a resting membrane potential that deviates from the calculated Nernst potential for K + (especially at low concentrations) whereas glial cells, which contain nongated ion channels for only potassium, have a resting membrane potential that matches closely with the calculated Nernst potential for K + .

The small amount of sodium conductance in the resting skeletal muscle, or nerve membrane, results in the resting membrane potential being slightly positive or depolarized compared with the equilibrium potential for potassium ( Table 2 ). The specific class of potassium channel that determines the resting membrane potential is the inward or anomalous rectifier potassium channel. Resting calcium conductance is exceedingly small. Therefore, calcium does not contribute to the resting membrane potential.

Table 2 . Membrane potential under different conditions

Membrane stateDominant membrane conductanceMembrane potential
RestingK + Close to K + equilibrium potential, approximately −95 mV
Peak of action potentialNa + Close to Na + equilibrium potential, approximately 40 mV

During an action potential, Na + channels open and the dominant membrane conductance is that of Na + . Consequently, the membrane potential is approximately the same as the Na + equilibrium potential ( Table 2 ).


Other Issues

Conductance-based models for excitable cells are developed to help understand underlying mechanisms that contribute to action potential generation, repetitive firing and bursting (i.e., oscillatory patterns) and so on. In turn, these intrinsic characteristics affect behaviors in neuronal networks.

However, as the number of currents included in conductance-based models expands, it becomes more difficult to understand and predict the resulting model dynamics due to the increasing number of differential equations. For example, the original Hodgkin-Huxley model is a 4th order system of ODEs. Efforts have been made not only to capture the qualitative dynamics of conductance-based models (e.g., FitzHugh-Nagumo model) but also to reduce the complexity of the system (e.g., Kepler et al. 1992).

Mathematical distinctions in conductance-based models using dynamical system and bifurcation analyses are available. Details are described in Izhikevich (2007).


The Three-dimensional Structure of a Bacterial K + Channel Shows How an Ion Channel Can Work

The remarkable ability of ion channels to combine exquisite ion selectivity with a high conductance has long puzzled scientists. K + leak channels, for example, conduct K + 10,000-fold better than Na + , yet the two ions are featureless spheres with similar diameters (0.133 nm and 0.095 nm, respectively). A single amino acid substitution in the pore of a K + channel can result in a loss of ion selectivity and cell death. The normal selectivity cannot be explained by pore size, because Na + is smaller than K + . Moreover, the high conductance rate is incompatible with the channel's having selective, high-affinity K + -binding sites, as the binding of K + ions to such sites would greatly slow their passage.

The puzzle was solved when the structure of a bacterial K + channelwas determined by x-ray crystallography. The channel is made from four identical transmembrane subunits, which together form a central pore through the membrane (Figure 11-23). Negatively charged amino acids are concentrated at the cytosolic entrance to the pore and are thought to attract cations and repel anions, making the channel cation-selective. Each subunit contributes two transmembrane helices, which are tilted outward in the membrane and together form a cone, with its wide end facing the outside of the cell where K + ions exit the channel. The polypeptide chain that connects the two transmembrane helices forms a short α helix (the pore helix) and a crucial loop that protrudes into the wide section of the cone to form the selectivity filter. The selectivity loops from the four subunits form a short, rigid, narrow pore, which is lined by the carbonyl oxygen atoms of their polypeptide backbones. Because the selectivity loops of all known K + channels have similar amino acid sequences, it is likely that they form a closely similar structure. The crystal structure shows two K + ions in single file within the selectivity filter, separated by about 8 Å. Mutual repulsion between the two ions is thought to help move them through the pore into the extracellular fluid.

Figure 11-23

The structure of a bacterial K + channel. (A) Only two of the four identical subunits are shown. From the cytosolic side, the pore opens up into a vestibule in the middle of the membrane. The vestibule facilitates transport by allowing the K + ions to (more. )

The structure of the selectivity filter explains the exquisite ion selectivity of the channel. For a K + ion to enter the filter, it must lose almost all of its bound water molecules and interact instead with the carbonyl oxygens lining the selectivity filter, which are rigidly spaced at the exact distance to accommodate a K + ion. A Na + ion, in contrast, cannot enter the filter because the carbonyl oxygens are too far away from the smaller Na + ion to compensate for the energy expense associated with the loss of water molecules required for entry (Figure 11-24).

Figure 11-24

K + specificity of the selectivity filter in a K + channel. The drawing shows K + and Na + ions (A) in the vestibule and (B) in the selectivity filter of the pore, viewed in cross section. In the vestibule, the ions are hydrated. In the selectivity filter, (more. )

Structural studies of the bacterial K + channel have indicated how these channels may open and close. The loops that form the selectivity filter are rigid and do not change conformation when the channel opens or closes. In contrast, the inner and outer transmembrane helices that line the rest of the pore rearrange when the channel closes, causing the pore to constrict like a diaphragm at its cytosolic end (Figure 11-25). Although the pore does not close completely, the small opening that remains is lined by hydrophobic amino acid side chains, which block the entry of ions.

Figure 11-25

A model for the gating of a bacterial K + channel. The channel is viewed in cross section. To adopt the closed conformation, the four inner transmembrane helices that line the pore on the cytosolic side of the selectivity filter (see Figure 11-22) rearrange (more. )

The cells that make most use of ion channels are neurons. Before discussing how they do so, we must digress to review briefly how a typical neuron is organized.


SK Channels Regulate Resting Properties and Signaling Reliability of a Developing Fast-Spiking Neuron

Reliable and precise signal transmission is essential in circuits of the auditory brainstem to encode timing with submillisecond accuracy. Globular bushy cells reliably and faithfully transfer spike signals to the principal neurons of the medial nucleus of the trapezoid body (MNTB) through the giant glutamatergic synapse, the calyx of Held. Thus, the MNTB works as a relay nucleus that preserves the temporal pattern of firing at high frequency. Using whole-cell patch-clamp recordings, we observed a K + conductance mediated by small-conductance calcium-activated potassium (SK) channels in the MNTB neurons from rats of either sex. SK channels were activated by intracellular Ca 2+ sparks and mediated spontaneous transient outward currents in developing MNTB neurons. SK channels were also activated by Ca 2+ influx through voltage-gated Ca 2+ channels and synaptically activated NMDA receptors. Blocking SK channels with apamin depolarized the resting membrane potential, reduced resting conductance, and affected the responsiveness of MNTB neurons to signal inputs. Moreover, SK channels were activated by action potentials and affected the spike afterhyperpolarization. Blocking SK channels disrupted the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons and induced extra postsynaptic action potentials in response to presynaptic firing. These data reveal that SK channels play crucial roles in regulating the resting properties and maintaining reliable signal transmission of MNTB neurons.SIGNIFICANCE STATEMENT Reliable and precise signal transmission is required in auditory brainstem circuits to localize the sound source. The calyx of Held synapse in the mammalian medial nucleus of the trapezoid body (MNTB) plays an important role in sound localization. We investigated the potassium channels that shape the reliability of signal transfer across the calyceal synapse and observed a potassium conductance mediated by small-conductance calcium-activated potassium (SK) channels in rat MNTB principal neurons. We found that SK channels are tonically activated and contribute to the resting membrane properties of MNTB neurons. Interestingly, SK channels are transiently activated by calcium sparks and calcium influx during action potentials and control the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons.

Keywords: MNTB SK channel excitability potassium channel resting membrane potential transmission fidelity.

Copyright © 2017 the authors 0270-6474/17/3710738-10$15.00/0.

Figures

SK channels mediated STOCs. A…

SK channels mediated STOCs. A , STOC recordings in control and after bath…

Development change of STOCs. A…

Development change of STOCs. A , Representative STOC recordings in rat MNTB neurons…

Calcium sparks activated the transient…

Calcium sparks activated the transient SK current. A , STOC recordings in control…

Tonic SK current. A ,…

Tonic SK current. A , B , Depolarizing voltage steps (15 s) from…

Activation of SK current. A…

Activation of SK current. A , A slow voltage ramp (5 mV/s) evoked…

Effects of SK channels on…

Effects of SK channels on resting membrane properties of MNTB neurons. A ,…

Effects of SK channels on…

Effects of SK channels on responsiveness. A–D , Voltage responses to synaptic-like waveforms…

Activation of SK channels by…

Activation of SK channels by Ca 2+ influx through NMDA receptors. A ,…

Activation of SK channels during…

Activation of SK channels during action potential. A , A brief voltage step…

SK-channel activation was required for…

SK-channel activation was required for highly reliable signal transmission. A , Representative trace…


Contents

A balance of magnesium is vital to the well-being of all organisms. Magnesium is a relatively abundant ion in Earth's crust and mantle and is highly bioavailable in the hydrosphere. This availability, in combination with a useful and very unusual chemistry, may have led to its utilization in evolution as an ion for signaling, enzyme activation, and catalysis. However, the unusual nature of ionic magnesium has also led to a major challenge in the use of the ion in biological systems. Biological membranes are impermeable to magnesium (and other ions), so transport proteins must facilitate the flow of magnesium, both into and out of cells and intracellular compartments.

Chlorophyll in plants converts water to oxygen as O2. Hemoglobin in vertebrate animals transports oxygen as O2 in the blood. Chlorophyll is very similar to hemoglobin, except magnesium is at the center of the chlorophyll molecule and iron is at the center of the hemoglobin molecule, with other variations. [6] This process keeps living cells on earth alive and maintains baseline levels of CO2 and O2 in the atmosphere.

Human health Edit

Inadequate magnesium intake frequently causes muscle spasms, and has been associated with cardiovascular disease, diabetes, high blood pressure, anxiety disorders, migraines, osteoporosis, and cerebral infarction. [7] [8] Acute deficiency (see hypomagnesemia) is rare, and is more common as a drug side-effect (such as chronic alcohol or diuretic use) than from low food intake per se, but it can occur in people fed intravenously for extended periods of time.

The most common symptom of excess oral magnesium intake is diarrhea. Supplements based on amino acid chelates (such as glycinate, lysinate etc.) are much better-tolerated by the digestive system and do not have the side-effects of the older compounds used, while sustained-release dietary supplements prevent the occurrence of diarrhea. [ citation needed ] Since the kidneys of adult humans excrete excess magnesium efficiently, oral magnesium poisoning in adults with normal renal function is very rare. Infants, which have less ability to excrete excess magnesium even when healthy, should not be given magnesium supplements, except under a physician's care.

Pharmaceutical preparations with magnesium are used to treat conditions including magnesium deficiency and hypomagnesemia, as well as eclampsia. [9] Such preparations are usually in the form of magnesium sulfate or chloride when given parenterally. Magnesium is absorbed with reasonable efficiency (30% to 40%) by the body from any soluble magnesium salt, such as the chloride or citrate. Magnesium is similarly absorbed from Epsom salts, although the sulfate in these salts adds to their laxative effect at higher doses. Magnesium absorption from the insoluble oxide and hydroxide salts (milk of magnesia) is erratic and of poorer efficiency, since it depends on the neutralization and solution of the salt by the acid of the stomach, which may not be (and usually is not) complete.

Magnesium orotate may be used as adjuvant therapy in patients on optimal treatment for severe congestive heart failure, increasing survival rate and improving clinical symptoms and patient's quality of life. [10]

Nerve conduction Edit

Magnesium can affect muscle relaxation through direct action on cell membranes. Mg 2+ ions close certain types of calcium channels, which conduct positively charged calcium ions into neurons. With an excess of magnesium, more channels will be blocked and nerve cells activity will decrease. [11] [12]

Hypertension Edit

Intravenous magnesium sulphate is used in treating pre-eclampsia. [13] For other than pregnancy-related hypertension, a meta-analysis of 22 clinical trials with dose ranges of 120 to 973 mg/day and a mean dose of 410 mg, concluded that magnesium supplementation had a small but statistically significant effect, lowering systolic blood pressure by 3–4 mm Hg and diastolic blood pressure by 2–3 mm Hg. The effect was larger when the dose was more than 370 mg/day. [14]

Diabetes and glucose tolerance Edit

Higher dietary intakes of magnesium correspond to lower diabetes incidence. [15] For people with diabetes or at high risk of diabetes, magnesium supplementation lowers fasting glucose. [16]

The U.S. Institute of Medicine (IOM) updated Estimated Average Requirements (EARs) and Recommended Dietary Allowances (RDAs) for magnesium in 1997. If there is not sufficient information to establish EARs and RDAs, an estimate designated Adequate Intake (AI) is used instead. The current EARs for magnesium for women and men ages 31 and up are 265 mg/day and 350 mg/day, respectively. The RDAs are 320 and 420 mg/day. RDAs are higher than EARs so as to identify amounts that will cover people with higher than average requirements. RDA for pregnancy is 350 to 400 mg/day depending on age of the woman. RDA for lactation ranges 310 to 360 mg/day for same reason. For children ages 1–13 years the RDA increases with age from 65 to 200 mg/day. As for safety, the IOM also sets Tolerable upper intake levels (ULs) for vitamins and minerals when evidence is sufficient. In the case of magnesium the UL is set at 350 mg/day. The UL is specific to magnesium consumed as a dietary supplement, the reason being that too much magnesium consumed at one time can cause diarrhea. The UL does not apply to food-sourced magnesium. Collectively the EARs, RDAs and ULs are referred to as Dietary Reference Intakes. [17]

Reference Daily Intake of magnesium [18]
Age Male Female Pregnancy Lactation
Birth to 6 months 30 mg* 30 mg*
7–12 months 75 mg* 75 mg*
1–3 years 80 mg 80 mg
4–8 years 130 mg 130 mg
9–13 years 240 mg 240 mg
14–18 years 410 mg 360 mg 400 mg 360 mg
19–30 years 400 mg 310 mg 350 mg 310 mg
31–50 years 420 mg 320 mg 360 mg 320 mg
51+ years 420 mg 320 mg

The European Food Safety Authority (EFSA) refers to the collective set of information as Dietary Reference Values, with Population Reference Intake (PRI) instead of RDA, and Average Requirement instead of EAR. AI and UL defined the same as in United States. For women and men ages 18 and older the AIs are set at 300 and 350 mg/day, respectively. AIs for pregnancy and lactation are also 300 mg/day. For children ages 1–17 years the AIs increase with age from 170 to 250 mg/day. These AIs are lower than the U.S. RDAs. [19] The European Food Safety Authority reviewed the same safety question and set its UL at 250 mg/day - lower than the U.S. value. [20] The magnesium UL is unique in that it is lower than some of the RDAs. It applies to intake from a pharmacological agent or dietary supplement only, and does not include intake from food and water.

For U.S. food and dietary supplement labeling purposes the amount in a serving is expressed as a percent of daily value (%DV). For magnesium labeling purposes 100% of the daily value was 400 mg, but as of May 27, 2016, it was revised to 420 mg to bring it into agreement with the RDA. [21] [22] Compliance with the updated labeling regulations was required by 1 January 2020, for manufacturers with $10 million or more in annual food sales, and by 1 January 2021, for manufacturers with less than $10 million in annual food sales. [23] [24] [25] During the first six months following the 1 January 2020 compliance date, the FDA plans to work cooperatively with manufacturers to meet the new Nutrition Facts label requirements and will not focus on enforcement actions regarding these requirements during that time. [23] A table of the old and new adult Daily Values is provided at Reference Daily Intake.

Green vegetables such as spinach provide magnesium because of the abundance of chlorophyll molecules, which contain the ion. Nuts (especially Brazil nuts, cashews and almonds), seeds (e.g., pumpkin seeds), dark chocolate, roasted soybeans, bran, and some whole grains are also good sources of magnesium. [26]

Although many foods contain magnesium, it is usually found in low levels. As with most nutrients, daily needs for magnesium are unlikely to be met by one serving of any single food. Eating a wide variety of fruits, vegetables, and grains will help ensure adequate intake of magnesium. [ citation needed ]

Because magnesium readily dissolves in water, refined foods, which are often processed or cooked in water and dried, in general, are poor sources of the nutrient. For example, whole-wheat bread has twice as much magnesium as white bread because the magnesium-rich germ and bran are removed when white flour is processed. The table of food sources of magnesium suggests many dietary sources of magnesium. [ citation needed ]

"Hard" water can also provide magnesium, but "soft" water contains less of the ion. Dietary surveys do not assess magnesium intake from water, which may lead to underestimating total magnesium intake and its variability.

Too much magnesium may make it difficult for the body to absorb calcium. [ citation needed ] Not enough magnesium can lead to hypomagnesemia as described above, with irregular heartbeats, high blood pressure (a sign in humans but not some experimental animals such as rodents), insomnia, and muscle spasms (fasciculation). However, as noted, symptoms of low magnesium from pure dietary deficiency are thought to be rarely encountered.

Following are some foods and the amount of magnesium in them: [27]

    seeds, no hulls (1/4 cup) = 303 mg , (1/4 cup) = 162 mg [28] flour (1/2 cup) = 151 mg (1/4 cup) = 125 mg
  • Oat bran, raw (1/2 cup) = 110 mg
  • Cocoa powder (1/4 cup) = 107 mg (3 oz) = 103 mg (1/4 cup) = 99 mg (1/4 cup) = 89 mg
  • Whole wheat flour (1/2 cup) = 83 mg , boiled (1/2 cup) = 79 mg , boiled (1/2 cup) = 75 mg , 70% cocoa (1 oz) = 73 mg , firm (1/2 cup) = 73 mg , boiled (1/2 cup) = 60 mg , cooked (1/2 cup) = 59 mg (2 tablespoons) = 50 mg (1/4 cup) = 46 mg , hulled (1/4 cup) = 41 mg , boiled (1/2 cup) = 39 mg , boiled (1/2 cup) = 37 mg , boiled (1/2 cup) = 36 mg , cooked (1/2 cup) = 32 mg (1 Tbsp) = 32 mg , non fat (1 cup) = 27 mg , espresso (1 oz) = 24 mg (1 slice) = 23 mg

In animals, it has been shown that different cell types maintain different concentrations of magnesium. [29] [30] [31] [32] It seems likely that the same is true for plants. [33] [34] This suggests that different cell types may regulate influx and efflux of magnesium in different ways based on their unique metabolic needs. Interstitial and systemic concentrations of free magnesium must be delicately maintained by the combined processes of buffering (binding of ions to proteins and other molecules) and muffling (the transport of ions to storage or extracellular spaces [35] ).

In plants, and more recently in animals, magnesium has been recognized as an important signaling ion, both activating and mediating many biochemical reactions. The best example of this is perhaps the regulation of carbon fixation in chloroplasts in the Calvin cycle. [36] [37]

Magnesium is very important in cellular function. Deficiency of the nutrient causes disease of the affected organism. In single-cell organisms such as bacteria and yeast, low levels of magnesium manifests in greatly reduced growth rates. In magnesium transport knockout strains of bacteria, healthy rates are maintained only with exposure to very high external concentrations of the ion. [38] [39] In yeast, mitochondrial magnesium deficiency also leads to disease. [40]

Plants deficient in magnesium show stress responses. The first observable signs of both magnesium starvation and overexposure in plants is a decrease in the rate of photosynthesis. This is due to the central position of the Mg 2+ ion in the chlorophyll molecule. The later effects of magnesium deficiency on plants are a significant reduction in growth and reproductive viability. [4] Magnesium can also be toxic to plants, although this is typically seen only in drought conditions. [41] [42]

In animals, magnesium deficiency (hypomagnesemia) is seen when the environmental availability of magnesium is low. In ruminant animals, particularly vulnerable to magnesium availability in pasture grasses, the condition is known as 'grass tetany'. Hypomagnesemia is identified by a loss of balance due to muscle weakness. [43] A number of genetically attributable hypomagnesemia disorders have also been identified in humans. [44] [45] [46] [47]

Overexposure to magnesium may be toxic to individual cells, though these effects have been difficult to show experimentally. [ citation needed ] Hypermagnesemia, an overabundance of magnesium in the blood, is usually caused by loss of kidney function. Healthy animals rapidly excrete excess magnesium in the urine and stool. [48] Urinary magnesium is called magnesuria. Characteristic concentrations of magnesium in model organisms are: in E. coli 30-100mM (bound), 0.01-1mM (free), in budding yeast 50mM, in mammalian cell 10mM (bound), 0.5mM (free) and in blood plasma 1mM. [49]

Mg 2+ is the fourth-most-abundant metal ion in cells (per moles) and the most abundant free divalent cation — as a result, it is deeply and intrinsically woven into cellular metabolism. Indeed, Mg 2+ -dependent enzymes appear in virtually every metabolic pathway: Specific binding of Mg 2+ to biological membranes is frequently observed, Mg 2+ is also used as a signalling molecule, and much of nucleic acid biochemistry requires Mg 2+ , including all reactions that require release of energy from ATP. [50] [51] [37] In nucleotides, the triple-phosphate moiety of the compound is invariably stabilized by association with Mg 2+ in all enzymatic processes.

Chlorophyll Edit

In photosynthetic organisms, Mg 2+ has the additional vital role of being the coordinating ion in the chlorophyll molecule. This role was discovered by Richard Willstätter, who received the Nobel Prize in Chemistry 1915 for the purification and structure of chlorophyll binding with sixth number of carbon

Enzymes Edit

The chemistry of the Mg 2+ ion, as applied to enzymes, uses the full range of this ion's unusual reaction chemistry to fulfill a range of functions. [50] [52] [53] [54] Mg 2+ interacts with substrates, enzymes, and occasionally both (Mg 2+ may form part of the active site). In general, Mg 2+ interacts with substrates through inner sphere coordination, stabilising anions or reactive intermediates, also including binding to ATP and activating the molecule to nucleophilic attack. When interacting with enzymes and other proteins, Mg 2+ may bind using inner or outer sphere coordination, to either alter the conformation of the enzyme or take part in the chemistry of the catalytic reaction. In either case, because Mg 2+ is only rarely fully dehydrated during ligand binding, it may be a water molecule associated with the Mg 2+ that is important rather than the ion itself. The Lewis acidity of Mg 2+ (pKa 11.4) is used to allow both hydrolysis and condensation reactions (most common ones being phosphate ester hydrolysis and phosphoryl transfer) that would otherwise require pH values greatly removed from physiological values.

Essential role in the biological activity of ATP Edit

ATP (adenosine triphosphate), the main source of energy in cells, must be bound to a magnesium ion in order to be biologically active. What is called ATP is often actually Mg-ATP. [5]

Nucleic acids Edit

Nucleic acids have an important range of interactions with Mg 2+ . The binding of Mg 2+ to DNA and RNA stabilises structure this can be observed in the increased melting temperature (Tm) of double-stranded DNA in the presence of Mg 2+ . [50] In addition, ribosomes contain large amounts of Mg 2+ and the stabilisation provided is essential to the complexation of this ribo-protein. [55] A large number of enzymes involved in the biochemistry of nucleic acids bind Mg 2+ for activity, using the ion for both activation and catalysis. Finally, the autocatalysis of many ribozymes (enzymes containing only RNA) is Mg 2+ dependent (e.g. the yeast mitochondrial group II self splicing introns [56] ).

Magnesium ions can be critical in maintaining the positional integrity of closely clustered phosphate groups. These clusters appear in numerous and distinct parts of the cell nucleus and cytoplasm. For instance, hexahydrated Mg 2+ ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes. [57]

Cell membranes and walls Edit

Biological cell membranes and cell walls are polyanionic surfaces. This has important implications for the transport of ions, in particular because it has been shown that different membranes preferentially bind different ions. [50] Both Mg 2+ and Ca 2+ regularly stabilize membranes by the cross-linking of carboxylated and phosphorylated head groups of lipids. However, the envelope membrane of E. coli has also been shown to bind Na + , K + , Mn 2+ and Fe 3+ . The transport of ions is dependent on both the concentration gradient of the ion and the electric potential (ΔΨ) across the membrane, which will be affected by the charge on the membrane surface. For example, the specific binding of Mg 2+ to the chloroplast envelope has been implicated in a loss of photosynthetic efficiency by the blockage of K + uptake and the subsequent acidification of the chloroplast stroma. [36]

Proteins Edit

The Mg 2+ ion tends to bind only weakly to proteins (Ka ≤ 10 5 [50] ) and this can be exploited by the cell to switch enzymatic activity on and off by changes in the local concentration of Mg 2+ . Although the concentration of free cytoplasmic Mg 2+ is on the order of 1 mmol/L, the total Mg 2+ content of animal cells is 30 mmol/L [58] and in plants the content of leaf endodermal cells has been measured at values as high as 100 mmol/L (Stelzer et al., 1990), much of which buffered in storage compartments. The cytoplasmic concentration of free Mg 2+ is buffered by binding to chelators (e.g., ATP), but also, what is more important, by storage of Mg 2+ in intracellular compartments. The transport of Mg 2+ between intracellular compartments may be a major part of regulating enzyme activity. The interaction of Mg 2+ with proteins must also be considered for the transport of the ion across biological membranes.

Manganese Edit

In biological systems, only manganese (Mn 2+ ) is readily capable of replacing Mg 2+ , but only in a limited set of circumstances. Mn 2+ is very similar to Mg 2+ in terms of its chemical properties, including inner and outer shell complexation. Mn 2+ effectively binds ATP and allows hydrolysis of the energy molecule by most ATPases. Mn 2+ can also replace Mg 2+ as the activating ion for a number of Mg 2+ -dependent enzymes, although some enzyme activity is usually lost. [50] Sometimes such enzyme metal preferences vary among closely related species: For example, the reverse transcriptase enzyme of lentiviruses like HIV, SIV and FIV is typically dependent on Mg 2+ , whereas the analogous enzyme for other retroviruses prefers Mn 2+ .

Importance in drug binding Edit

An article [59] investigating the structural basis of interactions between clinically relevant antibiotics and the 50S ribosome appeared in Nature in October 2001. High-resolution X-ray crystallography established that these antibiotics associate only with the 23S rRNA of a ribosomal subunit, and no interactions are formed with a subunit's protein portion. The article stresses that the results show "the importance of putative Mg 2+ ions for the binding of some drugs".

By radioactive isotopes Edit

The use of radioactive tracer elements in ion uptake assays allows the calculation of km, Ki and Vmax and determines the initial change in the ion content of the cells. 28 Mg decays by the emission of a high-energy beta or gamma particle, which can be measured using a scintillation counter. However, the radioactive half-life of 28 Mg, the most stable of the radioactive magnesium isotopes, is only 21 hours. This severely restricts the experiments involving the nuclide. Also, since 1990, no facility has routinely produced 28 Mg, and the price per mCi is now predicted to be approximately US$30,000. [60] The chemical nature of Mg 2+ is such that it is closely approximated by few other cations. [61] However, Co 2+ , Mn 2+ and Ni 2+ have been used successfully to mimic the properties of Mg 2+ in some enzyme reactions, and radioactive forms of these elements have been employed successfully in cation transport studies. The difficulty of using metal ion replacement in the study of enzyme function is that the relationship between the enzyme activities with the replacement ion compared to the original is very difficult to ascertain. [61]

By fluorescent indicators Edit

A number of chelators of divalent cations have different fluorescence spectra in the bound and unbound states. [62] Chelators for Ca 2+ are well established, have high affinity for the cation, and low interference from other ions. Mg 2+ chelators lag behind and the major fluorescence dye for Mg 2+ (mag-fura 2 [63] ) actually has a higher affinity for Ca 2+ . [64] This limits the application of this dye to cell types where the resting level of Ca 2+ is < 1 μM and does not vary with the experimental conditions under which Mg 2+ is to be measured. Recently, Otten et al. (2001) have described work into a new class of compounds that may prove more useful, having significantly better binding affinities for Mg 2+ . [65] The use of the fluorescent dyes is limited to measuring the free Mg 2+ . If the ion concentration is buffered by the cell by chelation or removal to subcellular compartments, the measured rate of uptake will give only minimum values of km and Vmax.

By electrophysiology Edit

First, ion-specific microelectrodes can be used to measure the internal free ion concentration of cells and organelles. The major advantages are that readings can be made from cells over relatively long periods of time, and that unlike dyes very little extra ion buffering capacity is added to the cells. [66]

Second, the technique of two-electrode voltage-clamp allows the direct measurement of the ion flux across the membrane of a cell. [67] The membrane is held at an electric potential and the responding current is measured. All ions passing across the membrane contribute to the measured current.

Third, the technique of patch-clamp uses isolated sections of natural or artificial membrane in much the same manner as voltage-clamp but without the secondary effects of a cellular system. Under ideal conditions the conductance of individual channels can be quantified. This methodology gives the most direct measurement of the action of ion channels. [67]

By absorption spectroscopy Edit

Flame atomic absorption spectroscopy (AAS) determines the total magnesium content of a biological sample. [62] This method is destructive biological samples must be broken down in concentrated acids to avoid clogging the fine nebulising apparatus. Beyond this, the only limitation is that samples must be in a volume of approximately 2 mL and at a concentration range of 0.1 – 0.4 μmol/L for optimum accuracy. As this technique cannot distinguish between Mg 2+ already present in the cell and that taken up during the experiment, only content not uptaken can be quantified.

Inductively coupled plasma (ICP) using either the mass spectrometry (MS) or atomic emission spectroscopy (AES) modifications also allows the determination of the total ion content of biological samples. [68] These techniques are more sensitive than flame AAS and are capable of measuring the quantities of multiple ions simultaneously. However, they are also significantly more expensive.

The chemical and biochemical properties of Mg 2+ present the cellular system with a significant challenge when transporting the ion across biological membranes. The dogma of ion transport states that the transporter recognises the ion then progressively removes the water of hydration, removing most or all of the water at a selective pore before releasing the ion on the far side of the membrane. [69] Due to the properties of Mg 2+ , large volume change from hydrated to bare ion, high energy of hydration and very low rate of ligand exchange in the inner coordination sphere, these steps are probably more difficult than for most other ions. To date, only the ZntA protein of Paramecium has been shown to be a Mg 2+ channel. [70] The mechanisms of Mg 2+ transport by the remaining proteins are beginning to be uncovered with the first three-dimensional structure of a Mg 2+ transport complex being solved in 2004. [71]

The hydration shell of the Mg 2+ ion has a very tightly bound inner shell of six water molecules and a relatively tightly bound second shell containing 12–14 water molecules (Markham et al., 2002). Thus, it is presumed that recognition of the Mg 2+ ion requires some mechanism to interact initially with the hydration shell of Mg 2+ , followed by a direct recognition/binding of the ion to the protein. [60] Due to the strength of the inner sphere complexation between Mg 2+ and any ligand, multiple simultaneous interactions with the transport protein at this level might significantly retard the ion in the transport pore. Hence, it is possible that much of the hydration water is retained during transport, allowing the weaker (but still specific) outer sphere coordination.

In spite of the mechanistic difficulty, Mg 2+ must be transported across membranes, and a large number of Mg 2+ fluxes across membranes from a variety of systems have been described. [72] However, only a small selection of Mg 2+ transporters have been characterised at the molecular level.

Ligand ion channel blockade Edit

Magnesium ions (Mg 2+ ) in cellular biology are usually in almost all senses opposite to Ca 2+ ions, because they are bivalent too, but have greater electronegativity and thus exert greater pull on water molecules, preventing passage through the channel (even though the magnesium itself is smaller). Thus, Mg 2+ ions block Ca 2+ channels such as (NMDA channels) and have been shown to affect gap junction channels forming electrical synapses.

The previous sections have dealt in detail with the chemical and biochemical aspects of Mg 2+ and its transport across cellular membranes. This section will apply this knowledge to aspects of whole plant physiology, in an attempt to show how these processes interact with the larger and more complex environment of the multicellular organism.

Nutritional requirements and interactions Edit

Mg 2+ is essential for plant growth and is present in higher plants in amounts on the order of 80 μmol g −1 dry weight. [4] The amounts of Mg 2+ vary in different parts of the plant and are dependent upon nutritional status. In times of plenty, excess Mg 2+ may be stored in vascular cells (Stelzer et al., 1990 [34] and in times of starvation Mg 2+ is redistributed, in many plants, from older to newer leaves. [4] [73]

Mg 2+ is taken up into plants via the roots. Interactions with other cations in the rhizosphere can have a significant effect on the uptake of the ion.(Kurvits and Kirkby, 1980 [74] The structure of root cell walls is highly permeable to water and ions, and hence ion uptake into root cells can occur anywhere from the root hairs to cells located almost in the centre of the root (limited only by the Casparian strip). Plant cell walls and membranes carry a great number of negative charges, and the interactions of cations with these charges is key to the uptake of cations by root cells allowing a local concentrating effect. [75] Mg 2+ binds relatively weakly to these charges, and can be displaced by other cations, impeding uptake and causing deficiency in the plant.

Within individual plant cells, the Mg 2+ requirements are largely the same as for all cellular life Mg 2+ is used to stabilise membranes, is vital to the utilisation of ATP, is extensively involved in the nucleic acid biochemistry, and is a cofactor for many enzymes (including the ribosome). Also, Mg 2+ is the coordinating ion in the chlorophyll molecule. It is the intracellular compartmentalisation of Mg 2+ in plant cells that leads to additional complexity. Four compartments within the plant cell have reported interactions with Mg 2+ . Initially, Mg 2+ will enter the cell into the cytoplasm (by an as yet unidentified system), but free Mg 2+ concentrations in this compartment are tightly regulated at relatively low levels (≈2 mmol/L) and so any excess Mg 2+ is either quickly exported or stored in the second intracellular compartment, the vacuole. [76] The requirement for Mg 2+ in mitochondria has been demonstrated in yeast [77] and it seems highly likely that the same will apply in plants. The chloroplasts also require significant amounts of internal Mg 2+ , and low concentrations of cytoplasmic Mg 2+ . [78] [79] In addition, it seems likely that the other subcellular organelles (e.g., Golgi, endoplasmic reticulum, etc.) also require Mg 2+ .

Distributing magnesium ions within the plant Edit

Once in the cytoplasmic space of root cells Mg 2+ , along with the other cations, is probably transported radially into the stele and the vascular tissue. [80] From the cells surrounding the xylem the ions are released or pumped into the xylem and carried up through the plant. In the case of Mg 2+ , which is highly mobile in both the xylem and phloem, [81] the ions will be transported to the top of the plant and back down again in a continuous cycle of replenishment. Hence, uptake and release from vascular cells is probably a key part of whole plant Mg 2+ homeostasis. Figure 1 shows how few processes have been connected to their molecular mechanisms (only vacuolar uptake has been associated with a transport protein, AtMHX).

The diagram shows a schematic of a plant and the putative processes of Mg 2+ transport at the root and leaf where Mg 2+ is loaded and unloaded from the vascular tissues. [4] Mg 2+ is taken up into the root cell wall space (1) and interacts with the negative charges associated with the cell walls and membranes. Mg 2+ may be taken up into cells immediately (symplastic pathway) or may travel as far as the Casparian band (4) before being absorbed into cells (apoplastic pathway 2). The concentration of Mg 2+ in the root cells is probably buffered by storage in root cell vacuoles (3). Note that cells in the root tip do not contain vacuoles. Once in the root cell cytoplasm, Mg 2+ travels toward the centre of the root by plasmodesmata, where it is loaded into the xylem (5) for transport to the upper parts of the plant. When the Mg 2+ reaches the leaves it is unloaded from the xylem into cells (6) and again is buffered in vacuoles (7). Whether cycling of Mg 2+ into the phloem occurs via general cells in the leaf (8) or directly from xylem to phloem via transfer cells (9) is unknown. Mg 2+ may return to the roots in the phloem sap.

When a Mg 2+ ion has been absorbed by a cell requiring it for metabolic processes, it is generally assumed that the ion stays in that cell for as long as the cell is active. [4] In vascular cells, this is not always the case in times of plenty, Mg 2+ is stored in the vacuole, takes no part in the day-to-day metabolic processes of the cell (Stelzer et al., 1990), and is released at need. But for most cells it is death by senescence or injury that releases Mg 2+ and many of the other ionic constituents, recycling them into healthy parts of the plant. In addition, when Mg 2+ in the environment is limiting, some species are able to mobilise Mg 2+ from older tissues. [73] These processes involve the release of Mg 2+ from its bound and stored states and its transport back into the vascular tissue, where it can be distributed to the rest of the plant. In times of growth and development, Mg 2+ is also remobilised within the plant as source and sink relationships change. [4]

The homeostasis of Mg 2+ within single plant cells is maintained by processes occurring at the plasma membrane and at the vacuole membrane (see Figure 2). The major driving force for the translocation of ions in plant cells is ΔpH. [82] H + -ATPases pump H + ions against their concentration gradient to maintain the pH differential that can be used for the transport of other ions and molecules. H + ions are pumped out of the cytoplasm into the extracellular space or into the vacuole. The entry of Mg 2+ into cells may occur through one of two pathways, via channels using the ΔΨ (negative inside) across this membrane or by symport with H + ions. To transport the Mg 2+ ion into the vacuole requires a Mg 2+ /H + antiport transporter (such as AtMHX). The H + -ATPases are dependent on Mg 2+ (bound to ATP) for activity, so that Mg 2+ is required to maintain its own homeostasis.

A schematic of a plant cell is shown including the four major compartments currently recognised as interacting with Mg 2+ . H + -ATPases maintain a constant ΔpH across the plasma membrane and the vacuole membrane. Mg 2+ is transported into the vacuole using the energy of ΔpH (in A. thaliana by AtMHX). Transport of Mg 2+ into cells may use either the negative ΔΨ or the ΔpH. The transport of Mg 2+ into mitochondria probably uses ΔΨ as in the mitochondria of yeast, and it is likely that chloroplasts take Mg 2+ by a similar system. The mechanism and the molecular basis for the release of Mg 2+ from vacuoles and from the cell is not known. Likewise, the light-regulated Mg 2+ concentration changes in chloroplasts are not fully understood, but do require the transport of H + ions across the thylakoid membrane.

Magnesium, chloroplasts and photosynthesis Edit

Mg 2+ is the coordinating metal ion in the chlorophyll molecule, and in plants where the ion is in high supply about 6% of the total Mg 2+ is bound to chlorophyll. [4] [83] [84] Thylakoid stacking is stabilised by Mg 2+ and is important for the efficiency of photosynthesis, allowing phase transitions to occur. [85]

Mg 2+ is probably taken up into chloroplasts to the greatest extent during the light-induced development from proplastid to chloroplast or etioplast to chloroplast. At these times, the synthesis of chlorophyll and the biogenesis of the thylakoid membrane stacks absolutely require the divalent cation. [86] [87]

Whether Mg 2+ is able to move into and out of chloroplasts after this initial developmental phase has been the subject of several conflicting reports. Deshaies et al. (1984) found that Mg 2+ did move in and out of isolated chloroplasts from young pea plants, [88] but Gupta and Berkowitz (1989) were unable to reproduce the result using older spinach chloroplasts. [89] Deshaies et al. had stated in their paper that older pea chloroplasts showed less significant changes in Mg 2+ content than those used to form their conclusions. The relative proportion of immature chloroplasts present in the preparations may explain these observations.

The metabolic state of the chloroplast changes considerably between night and day. During the day, the chloroplast is actively harvesting the energy of light and converting it into chemical energy. The activation of the metabolic pathways involved comes from the changes in the chemical nature of the stroma on the addition of light. H + is pumped out of the stroma (into both the cytoplasm and the lumen) leading to an alkaline pH. [90] [91] Mg 2+ (along with K + ) is released from the lumen into the stroma, in an electroneutralisation process to balance the flow of H + . [92] [93] [94] [95] Finally, thiol groups on enzymes are reduced by a change in the redox state of the stroma. [96] Examples of enzymes activated in response to these changes are fructose 1,6-bisphosphatase, sedoheptulose bisphosphatase and ribulose-1,5-bisphosphate carboxylase. [4] [53] [96] During the dark period, if these enzymes were active a wasteful cycling of products and substrates would occur.

Two major classes of the enzymes that interact with Mg 2+ in the stroma during the light phase can be identified. [53] Firstly, enzymes in the glycolytic pathway most often interact with two atoms of Mg 2+ . The first atom is as an allosteric modulator of the enzymes' activity, while the second forms part of the active site and is directly involved in the catalytic reaction. The second class of enzymes includes those where the Mg 2+ is complexed to nucleotide di- and tri-phosphates (ADP and ATP), and the chemical change involves phosphoryl transfer. Mg 2+ may also serve in a structural maintenance role in these enzymes (e.g., enolase).

Magnesium stress Edit

Plant stress responses can be observed in plants that are under- or over-supplied with Mg 2+ . The first observable signs of Mg 2+ stress in plants for both starvation and toxicity is a depression of the rate of photosynthesis, it is presumed because of the strong relationships between Mg 2+ and chloroplasts/chlorophyll. In pine trees, even before the visible appearance of yellowing and necrotic spots, the photosynthetic efficiency of the needles drops markedly. [73] In Mg 2+ deficiency, reported secondary effects include carbohydrate immobility, loss of RNA transcription and loss of protein synthesis. [97] However, due to the mobility of Mg 2+ within the plant, the deficiency phenotype may be present only in the older parts of the plant. For example, in Pinus radiata starved of Mg 2+ , one of the earliest identifying signs is the chlorosis in the needles on the lower branches of the tree. This is because Mg 2+ has been recovered from these tissues and moved to growing (green) needles higher in the tree. [73]

A Mg 2+ deficit can be caused by the lack of the ion in the media (soil), but more commonly comes from inhibition of its uptake. [4] Mg 2+ binds quite weakly to the negatively charged groups in the root cell walls, so that excesses of other cations such as K + , NH4 + , Ca 2+ , and Mn 2+ can all impede uptake.(Kurvits and Kirkby, 1980 [74] In acid soils Al 3+ is a particularly strong inhibitor of Mg 2+ uptake. [98] [99] The inhibition by Al 3+ and Mn 2+ is more severe than can be explained by simple displacement, hence it is possible that these ions bind to the Mg 2+ uptake system directly. [4] In bacteria and yeast, such binding by Mn 2+ has already been observed. Stress responses in the plant develop as cellular processes halt due to a lack of Mg 2+ (e.g. maintenance of ΔpH across the plasma and vacuole membranes). In Mg 2+ -starved plants under low light conditions, the percentage of Mg 2+ bound to chlorophyll has been recorded at 50%. [100] Presumably, this imbalance has detrimental effects on other cellular processes.

Mg 2+ toxicity stress is more difficult to develop. When Mg 2+ is plentiful, in general the plants take up the ion and store it (Stelzer et al., 1990). However, if this is followed by drought then ionic concentrations within the cell can increase dramatically. High cytoplasmic Mg 2+ concentrations block a K + channel in the inner envelope membrane of the chloroplast, in turn inhibiting the removal of H + ions from the chloroplast stroma. This leads to an acidification of the stroma that inactivates key enzymes in carbon fixation, which all leads to the production of oxygen free radicals in the chloroplast that then cause oxidative damage. [101]


What is 'calcium conductance'? - Biology

An intercalated disc is an undulating double membrane separating adjacent cells in cardiac muscle fibers. Intercalated discs support synchronized contraction of cardiac tissue. They can easily be visualized by a longitudinal section of the tissue.
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Aspects of the topic intercalated-disc are discussed in the following places at Britannica. Assorted References cardiovascular system ( in cardiovascular system (anatomy): Wall of the heart ) . cell volume, mitochondria occupy about 25 percent and provide the necessary energy for contraction. To facilitate energy and calcium conductance in cardiac muscle.
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Key Words: intercalated discs • kindlin-2 • cardiomyopathy • integrin . Thus, the intercalated discs ensure proper function of the heart with efficient .
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. Mechanical and Electrical Junctions at the Intercalated Disc . Gene Mutations, Defects in Intercalated Disc Proteins, and Arrhythmias. Top. Introduction .
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Intercalated disc information including symptoms, causes, diseases, symptoms, treatments, and other medical and health issues.
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What is intercalated disks? Meaning of intercalated disks medical term. What does . intercalated. intercalated disk. intercalated disks. intercalated .
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What is Intercalated disc? Meaning of Intercalated disc medical term. What does . intercalated disk (redirected from Intercalated disc) Also found in: .
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Myocyte intercalated discs were analysed by transmission electron microscope and . Intercalated discs were first photographed . Intercalated discs findings .
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2.3 Intercalated discs. 3 Role of calcium in contraction. 4 Regeneration of . Under electron microscopy, an intercalated disc's path appears more complex. .
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Intercalated,disc,biological ,biology dictionary,biology terminology,biology terms,biology abbreviations . are joined by intercalated discs that relay each .
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The intercalated disc of adult cardiac muscle consists of three main junctional . This suggests that the intercalated disc was saturated with cadherin protein in .
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Stacked images of intercalated discs were traced with IPLab . Immunoblotting of intercalated disc-associated proteins in . intercalated disc .
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The nexus in the intercalated disc of the canine heart: quantitative data for an . of radiopotassium across intercalated disks of mammalian cardiac muscle. .
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What is the purpose of the intercalated discs in cardiac muscle At an intercalated disc, the cell membranes of two adjacent cardiac muscle cells are extensively .
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Medical definition for the term 'intercalated disc' . Medical Dictionary - 'Intercalated Disc' How to search: . intercalated disc. Type: Term. Definitions: .
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What is 'calcium conductance'? - Biology

Synaptic plasticity is the strengthening or weakening of synapses over time in response to increases or decreases in their activity. Plastic change also results from the alteration of the number of receptors located on a synapse. Synaptic plasticity is the basis of learning and memory, enabling a flexible, functioning nervous system. Synaptic plasticity can be either short-term (synaptic enhancement or synaptic depression) or long-term. Two processes in particular, long-term potentiation (LTP) and long-term depression (LTD), are important forms of synaptic plasticity that occur in synapses in the hippocampus: a brain region involved in storing memories.

Long-term potentiation and depression: Calcium entry through postsynaptic NMDA receptors can initiate two different forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD). LTP arises when a single synapse is repeatedly stimulated. This stimulation causes a calcium- and CaMKII-dependent cellular cascade, which results in the insertion of more AMPA receptors into the postsynaptic membrane. The next time glutamate is released from the presynaptic cell, it will bind to both NMDA and the newly-inserted AMPA receptors, thus depolarizing the membrane more efficiently. LTD occurs when few glutamate molecules bind to NMDA receptors at a synapse (due to a low firing rate of the presynaptic neuron). The calcium that does flow through NMDA receptors initiates a different calcineurin and protein phosphatase 1-dependent cascade, which results in the endocytosis of AMPA receptors. This makes the postsynaptic neuron less responsive to glutamate released from the presynaptic neuron.

Short-term Synaptic Enhancement and Depression

Short-term synaptic plasticity acts on a timescale of tens of milliseconds to a few minutes. Short-term synaptic enhancement results from more synaptic terminals releasing transmitters in response to presynaptic action potentials. Synapses will strengthen for a short time because of either an increase in size of the readily- releasable pool of packaged transmitter or an increase in the amount of packaged transmitter released in response to each action potential. Depletion of these readily-releasable vesicles causes synaptic fatigue. Short-term synaptic depression can also arise from post-synaptic processes and from feedback activation of presynaptic receptors.

Long-term Potentiation (LTP)

Long-term potentiation (LTP) is a persistent strengthening of a synaptic connection, which can last for minutes or hours. LTP is based on the Hebbian principle: “cells that fire together wire together. ” There are various mechanisms, none fully understood, behind the synaptic strengthening seen with LTP.

One known mechanism involves a type of postsynaptic glutamate receptor: NMDA (N-Methyl-D-aspartate) receptors. These receptors are normally blocked by magnesium ions. However, when the postsynaptic neuron is depolarized by multiple presynaptic inputs in quick succession (either from one neuron or multiple neurons), the magnesium ions are forced out and Ca 2+ ions pass into the postsynaptic cell. Next, Ca 2+ ions entering the cell initiate a signaling cascade that causes a different type of glutamate receptor, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, to be inserted into the postsynaptic membrane. Activated AMPA receptors allow positive ions to enter the cell.

Therefore, the next time glutamate is released from the presynaptic membrane, it will have a larger excitatory effect (EPSP) on the postsynaptic cell because the binding of glutamate to these AMPA receptors will allow more positive ions into the cell. The insertion of additional AMPA receptors strengthens the synapse so that the postsynaptic neuron is more likely to fire in response to presynaptic neurotransmitter release. Some drugs co-opt the LTP pathway this synaptic strengthening can lead to addiction.

Long-term Depression (LTD)

Long-term depression (LTD) is essentially the reverse of LTP: it is a long-term weakening of a synaptic connection. One mechanism known to cause LTD also involves AMPA receptors. In this situation, calcium that enters through NMDA receptors initiates a different signaling cascade, which results in the removal of AMPA receptors from the postsynaptic membrane. With the decrease in AMPA receptors in the membrane, the postsynaptic neuron is less responsive to the glutamate released from the presynaptic neuron. While it may seem counterintuitive, LTD may be just as important for learning and memory as LTP. The weakening and pruning of unused synapses trims unimportant connections, leaving only the salient connections strengthened by long-term potentiation.