How can different ion channels of the same type have different cell responses?

The NMDA receptor is an ion channel and contributes to synaptic plasticity and memory. It is said that calcium ion flux through the receptor is critical for this mechanism. However, there are other ion channels that let calcium ions into the cell, how come they don't contribute to synaptic plasticity (to my knowledge)?

  • Intracellular receptors are located in the cytoplasm of the cell and are activated by hydrophobic ligand molecules that can pass through the plasma membrane.
  • Cell-surface receptors bind to an external ligand molecule and convert an extracellular signal into an intracellular signal.
  • Three general categories of cell-surface receptors include: ion -channel, G- protein, and enzyme -linked protein receptors.
  • Ion channel -linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through.
  • G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein, which then interacts with either an ion channel or an enzyme in the membrane.
  • Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme.
  • integral protein: a protein molecule (or assembly of proteins) that is permanently attached to the biological membrane
  • transcription: the synthesis of RNA under the direction of DNA

Cell Surface Receptors

Cell surface receptors are transmembrane proteins embedded into the plasma membrane which play an essential role in maintaining communication between the internal processes within the cell and various types of extracellular signals.

Such extracellular signals include hormones, cytokines, growth factors, neurotransmitters, lipophilic signaling molecules such as prostaglandins, and cell recognition molecules. When any of these ligands bind to their corresponding receptor, a conformational change is triggered which initiates an intracellular signaling pathway. Note that each ligand has its own specific cell surface receptor.

Image: “A schematic of a transmembrane receptor E = extracellular space P = plasma membrane I = intracellular space” by Mouagip. License: CC BY-SA 3.0

Additionally, cell surface receptors are specific to individual cell types and thus are also known as cell-specific proteins. These receptors regulate a multitude of biological pathways required for cell growth, survival, differentiation, proliferation, as well as many other cellular processes. Cell surface receptors are responsible for most of the signaling in multicellular organisms.

Cell surface receptors have the following components/domains:

  • The extracellular domain which binds ligands and is exposed to the outer surface of the cell also known as the recognition domain
  • The membrane-spanning region made up of hydrophobic protein molecules
  • The intracellular domain which is in contact with the cytoplasm also known as the coupling domain

Several factors govern the properties of these domains, including the size and extent of the domains, which may vary according to the type of cell surface receptor.

What is a Transporter

Transporter refers to a transmembrane protein that transports ions across the cell membrane against the concentration gradient by active transport. Hence, transporters consume energy in the form of ATP for the movement of ions. In other words, with the use of energy, transporters can move ions thermodynamically uphill to a higher energy state. A transporter can be either a primary pump and a secondary pump. The primary pumps hydrolyze ATP. With hydrolyzation, the conformation of the transporter changes and become capable of translocating the previously-bound specific ions, releasing them in or out of the cell. Sodium-potassium ATPase is an example of a primary transporter and it is shown in figure 2.

Figure 2: Sodium-Potassium ATPase

The regulation of transporters is achieved by the internal concentration of ions. The secondary pumps transport ions. They are capable of transporting two different types of ions: one ion is transported along its gradient and the other is transported against the gradient. The movement of the first ion serves as the energy source in the movement of the second ion. Symporters and antiporters are the two types of transporters. In symporters, each type of ions moves in the same direction across the membrane. In antiporters, the two types of ions move in the opposite direction across the membrane. Sodium-potassium-chloride symporter is an example of a secondary transporter.

Interaction of tumour cells with their microenvironment: ion channels and cell adhesion molecules. A focus on pancreatic cancer

Cancer must be viewed as a ‘tissue’, constituted of both transformed cells and a heterogeneous microenvironment, the ‘tumour microenvironment’ (TME). The TME undergoes a complex remodelling during the course of multistep tumourigenesis, hence strongly contributing to tumour progression. Ion channels and transporters (ICTs), being expressed on both tumour cells and in the different cellular components of the TME, are in a strategic position to sense and mediate signals arising from the TME. Often, this transmission is mediated by integrin adhesion receptors, which are the main cellular receptors capable of mediating cell-to-cell and cell-to-matrix bidirectional signalling. Integrins can often operate in conjunction with ICT because they can behave as functional partners of ICT proteins. The role of integrin receptors in the crosstalk between tumour cells and the TME is particularly relevant in the context of pancreatic cancer (PC), characterized by an overwhelming TME which actively contributes to therapy resistance. We discuss the possibility that this occurs through integrins and ICTs, which could be exploited as targets to overcome chemoresistance in PC.

1. Introduction

Tumour biology can be understood only taking into account both the individual transformed cells and the ‘tumour microenvironment’ (TME). The TME is a complex array of cells and extracellular matrix (ECM) proteins that tumour cells construct during the course of multistep tumourigenesis [1] and strongly influences the behaviour and malignancy of the transformed cells. Moreover, the TME may change during tumour progression, hence it may differ (structurally and functionally) from the primary tumour to its metastases [2,3]. The TME greatly varies among cancers of different histogenesis. For example, in leukaemias, it is mainly represented by the bone marrow, with the complex array of stromal and vascular cells which constitute the bone marrow nike, where leukaemia stem cells reside [4]. In carcinomas, a clear distinction is made between the neoplastic cells, named as the ‘parenchyma’, and the TME, indicated as the ‘tumour stroma’. An active and overwhelming tumour stroma (in this case, addressed as ‘desmoplastic reaction’) characterizes some specific carcinomas, such as breast, prostate or pancreatic cancer (PC) [5]. In particular, the desmoplastic reaction is one of the histopathological and functional hallmarks of PC: histopathological analysis reveals the presence of dense collagen (types I and III) bundles associated with fibroblasts, with loss of basement integrity and invasion of malignant cells into the interstitial matrix with exposure of collagens. The ECM in PC also contains fibronectins, tenascin-C, laminins, mainly secreted by pancreatic stellate cells (PSCs), a cellular phenotype peculiar of PC [6]. The desmoplastic reaction in PC is associated with an abnormal vasculature with numerous circuitous small leaky blood vessels and capillaries [7]. On the whole, the desmoplastic reaction is one of the major contributors to PC malignancy (see below).

Taking into account the relevance of the tumour stroma, antineoplastic therapeutic strategies must be tuned to target the ‘cancer tissue’, e.g. not only tumour cells, but also the cellular constituents of the TME [8,9]. In this context, deciphering the role of ion channel and transporter (ICT) proteins in the crosstalk between the tumour cells and the various constituents of the TME merits particular attention, also from a therapeutic viewpoint.

In this review, we briefly describe the TME as well as ICTs present in the different cells of the TME. For molecular and functional description of ICTs, we refer to other papers [10]. Then, we focus on adhesion receptors of the integrin family, and on their functional interaction with ICT. Because most of these data have been reported elsewhere [11], in this review, we mainly focus on PC, where the TME drives tumour progression and resistance to therapy.

2. The tumour microenvironment and its ion channels and transporters profile

The TME comprises both cells (endothelial cells and their precursors, fibroblasts and specialized mesenchymal cells as well as cells of the innate and specific immunity) and the proteins of the ECM. The main ICTs expressed in the cells of the TME have been detailed in [11] and are summarized in table 1. We must remember the prevalent role of Ca 2+ -permeable channels (both voltage-dependent and non-selective channels of the TRP family) in endothelial cells (ECs reviewed in [18,19]). The electrochemical driving force for Ca 2+ entry is provided by Ca 2+ -dependent K + channels (KCa), and the cooperation between Ca 2+ -permeable channels and KCa can serve to sustain the Ca 2+ -dependent secretion of growth and vasodilating factors by ECs. Because many recent studies are revealing distinctive gene expression profiles and cell-surface markers of tumour-associated versus normal ECs [20], it is possible that ICT can contribute to determine such difference. Owing to the relevant role of ECs and tumour angiogenesis in tumour progression, ICTs could be exploited to develop novel anti-angiogenesis therapies to selectively target the ECs inside the cancer tissue.

Table 1. Examples of ion channels expressed by different cell types participating to the TME complexity.

Inside the TME, both ‘cancer-associated fibroblasts’ [21] and specialized mesenchymal cells, such as myofibroblasts or the PSCs [22], are present. Although cancer-associated fibroblasts and specialized mesenchymal cells are not malignant, in that they do not bear cancerogenic mutations, they can exhibit epigenetic changes, which affect their behaviour [23]. Moreover, they are actively secreting ECM proteins, behaving as the main determinant of the desmoplastic reaction. The latter, directly or through the creation of a hypoxic environment, regulates tumour progression and dictates therapy resistance [24].

Finally, the TME is densely infiltrated by cells of both the innate and adaptive arms of the immune system, whose exact role in controlling tumour progression is still debated [25,26]. Neutrophil ion channels (mainly TRPs, KCa and Cl – channels reviewed in [27]) are exploited to accomplish the antimicrobial activity which characterizes these cells of innate immunity. Macrophages express KIR channels, which are involved in cell adhesion, and in turn affect the Ca 2+ -dependent macrophage activation [28]. Macrophages also express P2X7 receptors, which mediate the release of lysosomal cathepsin [29]. This fact could contribute to ECM remodelling, with a strong impact on malignant progression. The complex array of ion channels which contribute to T lymphocyte activation has been thoroughly described [15,16]: a coordinated influx of Ca 2+ is indeed essential to trigger T lymphocyte activation, and an unique contingent of ion channels (including Kv1.3 and KCa3.1 K + channels) orchestrate the duration and intensity of the Ca 2+ signals. Moreover, the balance of these channel types constitutes a specific functional marker of activated lymphocytes, thus providing a possible novel therapeutic target [11].

The ECM of the TME comprises proper matrix proteins, a multitude of growth factors and cytokines that, more or less directly, affect malignant cells [30]. Type I collagen, fibronectin and thrombospondins are the ECM proteins which characterize the TME, with collagen I being the main determinant of the desmoplastic reaction. ECM proteins can affect tumour progression by controlling cell motility as well as by dictating tumour cell differentiation programmes [30]. The ECM of the TME is produced by both tumour and mesenchymal cells, which also modulate their ECM by secreting proteases [31]. TME remodelling can lead to the release of molecules sequestered in the ECM, such as the vascular endothelial growth factors [32], and many cleavage products of ECM proteins [2], which further control tumour angiogenesis and metastasis formation.

The crosstalk between the ECM and tumour cells, as well as of the cellular components of the TME, is mainly mediated through the intervention of adhesion receptors of the integrin family.

3. Cell adhesion molecules: the integrin family

(a) Integrin structure and function

Integrin receptors are transmembrane proteins formed by non-covalent association of α- and β-subunits. To date, 18 α- and eight β-subunits are known in mammals. All subunits are type I transmembrane glycoproteins with a short cytoplasmic tail (20–70 amino acids), a membrane-spanning helix and a large multidomain extracellular portion [33]. The β4-subunit is an exception because its cytoplasmic domain contains around 1000 amino acids [34]. Integrin subunits can combine to form at least 24 functional heterodimers, each of which binds a specific array of ECM proteins, or cell adhesion molecules (CAMs), that act as ‘counterreceptors’.

Integrins are more than CAMs: they can transmit bidirectional signals across the plasma membrane. On the cell surface, integrins are normally in the low affinity state, and they can be activated through an ‘inside-out’ signalling pathway. During this process, two cellular activators, talin and kindlin, play an essential role, and the binding of talin to the β integrin cytoplasmic tail is proposed to be the final step in integrin activation [35]. Conversely, the binding of the integrin extracellular ligands transmits signals inward, a process called ‘outside-in’ signalling. Outside-in signalling of integrin exerts significant influences on cell mobility, proliferation, differentiation, etc. [36]. Because the list of papers describing the many facets of integrin-mediated signalling pathways is immense, we refer to those [33,37,38], and only limit to summarize that integrins seem to be linked to almost all of the known signalling pathways, including induction of cytosolic kinases, stimulation of the phosphoinositide metabolism, activation of Ras/MAPK and protein kinase C (PKC) pathways and regulation of small GTPases. Moreover, integrin signalling often overlaps with that triggered by growth factor or cytokine receptors [38]. The overlap and proper integration of differently arising signals, makes physiological sense, because cells must integrate multiple stimuli from the ECM, growth factors, hormones and mechanical stress, to organize appropriate responses. The same integration occurs and determines the fate of even more in tumour cells inside the cancer tissue.

(b) Integrin relationships with ion channels

The relationships between integrins and ion channels in the cell-to-cell and cell-to-matrix interactions have been long described [39]. The earliest indications came from studies on neuronal and leukaemic cells, in which many cellular processes elicited by the engagement of adhesive proteins, such as differentiation, migration and neurite extension, turned out to depend on ion channel activation [40–43]. When associated with integrins, ion channel function becomes bidirectional itself: it is regulated by extracellular signals (through integrins) and in turn controls integrin activation and/or expression [39]. Interestingly, the same kind of complex bidirectional signalling has been observed for some ion transporters, in particular those mediating proton fluxes [44–46], which are so relevant in the establishment of a reversed H + gradient which characterizes neoplastic malignancy [47].

The bidirectional crosstalk between integrins and ion channels occurs through different mechanisms: it may rely on cytoplasmic messengers, such as Ca 2+ or protein kinases, commuting between the two proteins (reviewed in [39]). For example, T lymphocyte activation in which β1 integrins are involved is underlied by a coordinated influx of Ca 2+ , which is controlled by and, conversely, regulates K + channels [16]. The transmission of mechanical forces at focal adhesion sites is triggered by integrins and mediated by calcium [48], but also involves the activation of signalling molecules, such as FAK and c-Src [49].

Another aspect of integrin/ion channel interaction is the fact that integrins and ion channels can interact directly at the plasma membrane level. In other words, the two proteins co-assemble on the plasma membrane and give rise to supramolecular complexes, which constitute platforms for triggering and orchestrating downstream intracellular signals. The first evidence was obtained in immune cells by Levite et al. [50], who found that the β1 integrin subunit associated with Kv1.3 channels in T lymphocytes. Shortly afterwards, a physical link between Kv1.3 channels and β1 integrins was described in melanoma cells [51]. Our group found that the β1 integrin subunit associates with another K + channel, Kv 11.1 or hERG1, on the plasma membrane of tumour cells, either leukaemias or solid cancers [52–56]. This complex can also involve growth factor or chemokine receptors and, once assembled, recruits cytosolic signalling proteins, which in turn activate intracellular signalling in an integrin- and ion channel-dependent manner. This has a clear negative impact on the leukaemia disease [54], can trigger chemoresistance [55] or control angiogenesis and tumour progression [56].

Another mechanism involving the interaction between integrins and ion channels contributes to determine integrin recycling [57]. In particular, CLIC3 chloride channels colocalize with active α5β1 integrins in late endosomes/lysosomes, allowing the integrin to be retrogradely transported and recycled to the plasma membrane at the cell rear. This mechanism also involves Rab25 and has a clear impact on cancer behaviour. In fact, in PC, active integrins and CLIC3 are necessary for cancer cell invasion [57].

(c) Integrins and ion channels: role in cell migration

A most interesting aspect regards the comprehension of several mechanisms by which integrins and different channel types interact in controlling cell migration. Besides being a fundamental component of embryogenesis and tissue remodelling in the adult, these processes are relevant in tumour cell invasiveness and metastatic spread. As typical mediators of cell interaction with the environment, it is not surprising that integrins play major roles in eukaryotic cell migration. Moreover, we are nowadays aware that several types of Ca 2+ -activated and voltage-dependent K + channels are also implicated in the cell migration machinery. This rapidly growing field has been reviewed recently [58,59] and will not be discussed in detail here. We limit our discussion to the fact that K + channels can form complexes, and thereby modulate several proteins involved in cell movement, such as FAK [60,61], cortactin [62,63] and integrins themselves. Interesting speculations can derive from studies on α9β1 integrins, which can regulate cell movement by activating inward rectifier K + channels (IRK) [64]. IRK channels, along with the integrin, are physically linked to spermidine/xspermine N1-acetyltransferase, the key enzyme in the pathway that acetylates spermine and spermidine to putrescine, thus controlling the intracellular concentration of polyamines. Polyamines are critical regulators of neoplastic growth and also the main intracellular messengers controlling IRK activity. A functional network may hence be figured out, where an adequate intracellular concentration of polyamines converges to trigger a proper α9β1-dependent cell movement, through the modulation of IRK channels [65].

(d) Integrins and ion channels in the cells of the tumour microenvironment

Integrins and ion channels also interact at the level of the TME. One example involves cells of the innate immune system: neutrophils release Cl – to accomplish their antimicrobial activity Cl – release occurs through the activation of Cl – channels which is, at least in part, dependent on β2 integrin-mediated adherence to fibronectin [66]. Macrophages express KIR channels, whose activity is modulated by VLA4 (α4β1) integrin receptors and hence by cell adhesion, which in turn affects the Ca 2+ -dependent macrophage activation [28]. Another example is represented by ECs and their Cl channels of the CLCA protein family. In ECs, CLCA2 behaves as a vascular addressin for metastatic, blood-borne, cancer cells, facilitating vascular arrest of cancer cells via adhesion to β4 integrins, and hence promoting metastatic spread. In addition, the β4-integrin–CLCA complex stimulates Src-dependent cell signalling through FAK and extracellular signal-regulated kinase (ERK), leading to increased proliferation of metastatic foci [67].

A list of ion channels physically or functionally linked to integrins in the cells of TME is reported in table 2.

Table 2. Examples of interactions between ion channels and integrin subunits in cells of TME.

4. The role of the tumour microenvironment in the progression of pancreatic cancer

PC, mainly represented by the histological form of pancreatic ductal adenocarcinoma, is one of the most lethal gastrointestinal malignancies, representing the second leading cause of death among them. The overall 5-year survival rate is less than 6%, in the most recent American Database (, and a median survival of 18 months from diagnosis for those operated-on patients with no evidence of residual disease. Similar disappointing figures are available from European surveys [69]. The malignant nature of PC is mainly due to its aggressive growth and rapid development of distant metastases, thus making treatment extremely difficult. Additionally, PC is locally invasive, surrounded by a dense desmoplastic reaction (see §1) which can involve adjacent vital structures, limiting the chance for complete resection. Indeed, less than 20% of patients are candidates for surgical resection at the time of diagnosis, while almost one half have metastatic disease. Moreover, of the few patients who undergo surgery with radical intent (R0), most will develop a recurrence within 15 months. Although surgery remains the cornerstone of cure, the addition of adjuvant treatments is required [70]. Chemotherapy (in Europe) or radiochemotherapy (in North America) has been used, either as adjuvant to surgery or as definitive treatment for unresectable disease, with conflicting results [71,72]. Failure of traditional therapeutic approaches for this devastating disease had led to many efforts towards the study of molecular biology and targeted therapies, in order to create a multimodal therapeutic strategy [73]. Among target therapy drugs, only erlotinib that targets the EGF-R, has been shown to improve survival when used in combination with gemcitabine, compared with gemcitabine alone. Nevertheless, the clinical response rate remains modest, mainly owing to the intrinsic chemoresistance of PC cells [74]. Investigating mechanisms mediating chemoresistance is therefore of clinical interest in drug development of new agents. As stated in §1 one of the major contributors to PC malignancy and therapy resistance is represented by the desmoplastic reaction. Further understanding of how the TME facilitates PC cell malignancy will identify unique targets that may finally improve the treatment of patients with PC [6]. Some insights are briefly reported below.

(a) Interaction between tumour and stromal cells in pancreatic cancer: the role of pancreatic stellate cells

Normal development of glandular structures requires interactions between stromal cells and the epithelial cells that will eventually line the surface of the gland. In the pancreas, mesenchymal cells stimulate adjacent pluripotent cells to form acini, while inducing other remote cells to complete the endocrine pathway. These interactions in normal development require specific proteins within the ECM, notably laminin. Crosstalk between mesenchymal and epithelial compartments occurs through soluble messengers acting on a paracrine (or autocrine) manner, cell-surface receptor activation through direct cell-to-cell contact, and specific ECM proteins secreted by mesenchymal cells [75,76]. Perturbations of the normal mesenchymal–epithelial interactions can lead to unregulated cell growth, as occurs in cancer.

PSCs are the predominant mesenchymal cells within the PC stroma and the main determinant of the desmoplastic reaction [77]. PSCs originate from bone-marrow-derived mesenchymal stem cells, and are similar to myofibroblasts found in other tumour stroma (breast, prostate cancer). PSCs have the ability to transdifferentiate from a ‘quiescent’, retinoid/lipid storing phenotype in the normal pancreas to an ‘activated’, α-smooth muscle actin producing myofibroblastic phenotype. Activators of PSCs in vivo include cytokines (IL1, IL6, IL8 and TNF-α) growth factors (PDGF and TGF-β) and reactive oxygen species released by damaged inflammatory cells recruited in response to injury to the pancreas. Activated PSCs, in turn, can produce autocrine factors, such as PDGF, TGF-β, cytokines and proinflammatory molecules which may potentiate an activated phenotype [77–79]. PSCs, besides being implicated in the genesis of chronic pancreatitis, are critical for the development of the desmoplastic reaction in PC, being the main source of ECM proteins [78]. Moreover, because PSCs are activated by PC cells in vitro, a synergistic relationship between the two types of cells occurs, that favours the development and progression of PC.

(b) Integrins in pancreatic cancer

In PC, it is well established that ECM proteins, such as collagen, fibronectin and tenascin-C, interact with cell-surface integrins to provide intracellular signals to both PSCs and PC cells [80]. For example, in PSCs, β1 integrins are important not only to modulate cell adhesion to the basement membrane, but also to determine the proper formation and differentiation of acini during normal development [81]. PC cells express at least two major classes of integrins: β1-containing integrins (mainly α2β1 and α5β1), which mediate cell adhesion to fibronectin, laminin and collagen (type I and IV), and αvβ5, which is mainly involved in PC cell adhesion to vitronectin [82,83]. Integrin engagement has been reported to determine the malignant phenotype of PC cells by regulating cell proliferation [82], invasion [84] as well as by cytokine secretion [85]. For example, tenascin-C can affect PC cell growth and migration through the activation of β1 integrin intracellular signalling pathways [86]. Some recent evidence indicates that intergrin involvement in PC cell invasion can also occur through a complex interaction with EGF receptor (EGF-R) and the modulation of Src-centred intracellular signalling pathways [84].

On the whole, integrins, mainly those containing the β1 subunit, being expressed both in PC cells and in PSCs, and regulating the production of ECM proteins by the latter, are pivotal in regulating tumour–stroma interaction in PC. β1 integrins can hence drive different aspects of tumour cell behaviour, including chemo- and radio-resistance (see below).

(c) Ion channels and transporters in pancreatic cancer

In the past few years, different ICT proteins have been characterized in PC cells: TRP cationic channels of the ‘melastatin-related’ type (TRPM) have been reported to be overexpressed in PC cells. In particular, both TRPM8 [87] and TRPM7 [88] are overexpressed in pancreatic ductal adenocarcinoma cells, where they regulate either cell proliferation or migration. TRPM7 activation triggers a Mg 2+ -sensitive ‘suppressor of cytokine signalling 3a’ (Socs3a) pathway, which regulates exocrine pancreatic epithelial cell proliferation, both in development and cancer [89]. Moreover, Dong et al. [90] reported that TGF-β induces Ca 2+ entry in PC cells via TRPC1 channels and the Na + /Ca 2+ exchanger NCX1, thus raising intracellular Ca 2+ concentration. Ca 2+ increase is in turn essential for PKCα activation and subsequent tumour cell invasion [90]. As described above [57], CLIC3 channels regulate integrin recycling and behave as independent prognostic indicators in PC. This highlights the importance of active integrin trafficking as well as Cl – channels as potential drivers to cancer progression. Among voltage-gated K + channels, Kv 11.1 (hERG1) is expressed in PC and identifies a patient group with worse prognosis [91]. Because hERG1 physically and functionally interacts with β1 integrins in different tumour cells (see above) including PC [91], it is tempting to speculate that it could drive PC malignancy through the modulation of integrin-mediated signalling. Finally, the sodium hydrogen exchanger 1 (HNE1) is expressed in PC cells [92,93] and is activated by growth factors such as neurotensin (NT). The NT/HNE1 pathway may be implicated in the early progression of PC by localized acidification and induction of an aerobic glycolytic phenotype with higher metastatic potential.

A model illustrating tumour–stroma interaction in PC, as well as the involvement of integrins, is reported in figure 1. Those ICTs which interact with integrins to modulate the crosstalk between PC cells and the TME are also illustrated.

Figure 1. A model illustrating tumour–stroma interaction in PC as well as the involvement of integrins.

(d) The tumour microenvironment drives therapy resistance in pancreatic cancer

The altered crosstalk between stromal ECM proteins and integrins expressed on both tumour and TME cells is also implicated in mechanisms of acquired resistance to chemo- and radiotherapy in PC. An in vitro study using different PC cell lines cultured in the presence of collagen, fibronectin or laminin, showed different induction of chemosensitivity, depending on the type of substrate [94]. Indeed, inhibiting ECM-integrin function in combination with chemotherapy may be a potential therapeutic intervention that could specifically target the desmoplastic reaction. One example could be the monoclonal antibody targeting α5β1 or αvβ3 integrin (Vitaxin). Moreover, PSCs are known to promote radioprotection of PC cells: this effect is dependent on a signalling pathway triggered by β1 integrin and converging on FAK [95].

Treatment failure in PC may be due, at least in part, to our limited understanding of how the fibrotic tissue and the stromal cells present within this tumour can facilitate the rapid progression of this cancer type. Therefore, a better understanding of the mechanisms which regulate PC cell interactions with the TME, with particular attention to the crosstalk between integrins and ICTs, could open a new perspective to effectively treat PC.

Ion Channel Involvement in Desmoplasia

Fibrosis is a pathological outcome common for many chronic inflammatory diseases including chronic pancreatitis (Wynn and Ramalingam, 2012). The abundant stroma reaction (desmoplasia) is a hallmark common to both chronic pancreatitis and PDAC (Haeberle et al., 2018). Chronic pancreatitis is considered a risk factor for pancreatic cancer, and indeed, it frequently evolves to a true PDAC (McKay et al., 2008). In both cases, the normal pancreatic parenchyma is markedly remodeled (as shown in Figure 1) so that the normal organ function is eventually lost. The poorly vascularized desmoplastic tissue is characterized by high stiffness, low elasticity, and high tissue pressure (up to 100 mmHg) (Stylianopoulos et al., 2012 Fels et al., 2016 Pethő et al., 2019), which leads to impaired perfusion of the tumor tissue with the further result of tissue hypoxia. The pancreatic stellate cells (PSCs) are believed to be the key effectors behind the stroma deposition in PDAC and chronic pancreatitis (Haeberle et al., 2018). Desmoplasia represents an important challenge that new PDAC therapies have to deal with (Henke et al., 2020). The absence of vascularization combined with vessel compression because of the massive fibrosis prevents the efficient delivery of the chemotherapeutic drugs (Dauer et al., 2018).

FIGURE 1. (A) Histomorphology of a healthy human pancreas, hematoxylin and eosin (Hɮ). The parenchymal structure of the organ is clearly visible. Acinar cells are identifiable by their typical round shape. Their bases are stained in blue due to the presence of the nuclei, while their apices are pink due to the high concentration of zymogen. Two islets are located in the central and right parts of the image. The cytoplasm of the islet cells is paler than the surrounding acinar cells. (B) Histomorphology of a chronic pancreatitis, hematoxylin and eosin (Hɮ). The tissue is characterized by an evident increase in interlobular fibrosis, atrophy of the acini, and inflammatory infiltrate, which is evident when compared to the healthy component of the same sample (inset). (C,D) Histomorphology of two human pancreatic ductal adenocarcinomas (PDACs), hematoxylin and eosin (Hɮ). The normal architecture of the parenchyma is lost. Multiple layers of cells highlight the neoplastic lesions in panel (C). High levels of desmoplasia (colored in pink) are present especially in panel (D). Distribution of different cell populations is detectable in the tumor tissue neoplastic cells (pointed by black arrows) are embedded in a dense desmoplastic stroma (pointed by yellow arrows). Evident immune cells infiltration (pointed by red arrows) is present on the right side of the figure. Immune cells are identifiable by their small sizes and the intense basophilic staining of the nuclei. Scale bars: 100 μm.

Consequently, new strategies targeting the stroma compartment have emerged. This includes the attempt to attenuate/reverse the activation of the cancer-associated fibroblasts (CAFs) which also includes PSCs. The results of these studies however are contradictory. Inhibiting the TGF-β signaling pathway with the anticancer compound Minnelide, which is able to reverse the activation state of the CAFs, has a similar positive effect in a murine PDAC model (Dauer et al., 2018) as the inhibition of hedgehog signaling in CAFs with IPI-926. Moreover, IPI-926 also increases the delivery and the efficacy of gemcitabine in mice (Olive et al., 2011). However, other studies highlighted that an uncontrolled depletion of the stroma compartment rather promotes PDAC progression than slowing it down (Özdemir et al., 2014). Consequently, the understanding of the stromal compartment in PDAC has to be further refined. It has become apparent that cancer-associated fibroblasts constitute a heterogeneous cell population with distinct gene expression profiles, location within the tumor, and function (Von Ahrens et al., 2017 Öhlund et al., 2017 Miyai et al., 2020). Öhlund et al. propose a distinction between inflammatory fibroblasts, mainly responsible for the secretion of inflammatory factors, and myofibroblasts that are responsible for the ECM production (Öhlund et al., 2017). PSCs are included in this last category. To the best of our knowledge, it has not yet been studied whether these two types of CAFs are also equipped with distinct sets of ion channels.

Pancreatic Stellate Cells

In a healthy pancreas, PSCs are usually kept in a quiescent state, and they are responsible for the maintenance of the tissue integrity by regulating the ECM turnover (Haeberle et al., 2018). In PDAC, PSCs become strongly activated by the secretome and the physicochemical properties of the PDAC microenvironment (Omary et al., 2007a). Thus, PSCs are activated among others by inflammatory mediators, growth factors (PDGF and TGF-㬡), cytokines (IL-1, IL-6, and IL-8), hormones, angiotensin II, intracellular signaling molecules, and reactive oxygen species (ROS) as well as hypoxia (Nielsen et al., 2017) and mechanical stimuli (Omary et al., 2007b Fels et al., 2016, Fels et al., 2018 Ferdek and Jakubowska, 2017 Lachowski et al., 2017). Activated PSCs, in turn, secrete growth factors themselves so that they are engaged in a mutual positive feedback loop of other cells of the PDAC tissue (Fu et al., 2018). In addition, activated PSCs proliferate, migrate (Omary et al., 2007b), and secrete copious amounts of ECM components, especially collagen I and III (Ferdek and Jakubowska, 2017). The resulting changes in the pH values and increased stiffness of the desmoplastic tissue also feed back onto the behavior of PSCs (Lachowski et al., 2017). One of the mechanosensitive ion channels, Piezo1, that senses the mechanical properties of the PDAC microenvironment is inhibited by an acidic pH. This could prevent PSCs to be overridden by the mechanically triggered Ca 2+ influx via Piezo1 channels (Kuntze et al., 2020).

Ion Channels and Fibrosis

The function of ion channels in tumor stroma cells is far from being fully understood, especially regarding PDAC. Nonetheless, we already know that some ion channels play a significant role in the development of fibrosis in other organs such as KCa3.1 in lungs, kidneys (Roach et al., 2013), and heart (Zhao et al., 2015) K2P2.1 in cardiac fibrosis (Abraham et al., 2018) and TRPV4 in liver (Songa et al., 2014), heart (Adapala et al., 2013), and lung fibrosis (Rahaman et al., 2014). Usually the inhibition of these ion channels attenuates the profibrotic response of the fibroblasts (Cruse et al., 2011 Adapala et al., 2013 Rahaman et al., 2014 Abraham et al., 2018 Roach and Bradding, 2019).

Ion channel research in PSCs is still in its infancy. We will therefore draw some analogies from hepatic stellate cells that are closely related to PSCs and in which these ion channels may play a similar role. Hepatic stellate cells are responsible for matrix homeostasis in healthy livers (Puche et al., 2013). Similar to the PSCs in PDAC, they are mainly responsible for the excessive production and remodeling of the ECM in the fibrotic liver (Puche et al., 2013 Freise et al., 2015 Ezhilarasan et al., 2018). For this reason, these types of cells have been suggested as a possible target for antifibrotic therapy.

KCa3.1: We do not have much information on the role of KCa3.1 channels in PDAC-associated fibrosis, which is largely driven by PSCs. It is only known that KCa3.1 channels regulate migration of PSCs (Storck et al., 2017).

So far, it is under debate whether KCa3.1 has pro- or antifibrotic effects in the liver (Roach and Bradding, 2019). KCa3.1 expression is increased in hepatic stellate cells after the incubation with TGF-β, a known activator of hepatic stellate cells. In both in vitro and in vivo experiments, the inhibition of KCa3.1 shows an antifibrotic effect and decreases the expression of profibrotic genes (Freise et al., 2015). On the contrary, in the work of Møller et al., the inhibition or the absence of KCa3.1 in hepatic stellate cells and hepatocytes worsens liver fibrosis (Møller et al., 2016). This information highlights the possible problems that ion channel therapies could face the inhibition of an ion channel expressed in different cell types could have different effects.

K2P2.1: So far, we only know that PSCs express K2P2.1 (previously designated as TWIK-related potassium channel-1 TREK1) (Fels et al., 2016). In fact, K2P2.1 is a mechanosensitive ion channel that can be modulated by pressure and membrane stretch (Lauritzen et al., 2005 Honoré, 2007) but also by pH. K2P2.1 contributes to setting the resting membrane potential of the cells (Bittner et al., 2014), and it is strongly correlated with proliferation and cell cycle in some tumors (Pethő et al., 2019). The mechanosensitive function of K2P2.1 is postulated to be involved in the migration, especially in the coordination of the front and rear ends of the cells (Pethő et al., 2019). Sauter et al. observed that the activation of K2P2.1 with BL 1249 in a PDAC line, BxPC-3, inhibits cell proliferation and migration through the hyperpolarization of the membrane (Sauter et al., 2016). Controversially, the absence of K2P2.1 in heart myofibroblasts from pressure-overloaded mice attenuates cardiac fibrosis also by decreasing fibroblast proliferation and migration (Abraham et al., 2018). This highlights again how the same ion channel can have a different impact on the behavior of different cell types and how this topic must be considered during the development of new therapies. However, it may also be seen as an indication that the “natural,” possibly fluctuating, activity is what matters physiologically. Clamping channel activity to a maximum or a minimum impairs cell function. It remains to be seen whether K2P2.1 channels exert a similar role in PDAC desmoplasia, where the unique tumor microenvironment could influence K2P2.1 function in many ways.

TRPV4: The transient receptor potential vanilloid channel 4 (TRPV4) is a mechanosensitive Ca 2+ -permeable nonselective cation channel that is expressed in many organs including the pancreas (Zhan and Li, 2018). TRPV4 is also expressed in PSCs. Its mRNA expression strongly decreases in PSCs when they are cultured under an elevated ambient pressure (𫄀 mmHg), mimicking the conditions that can be found in PDAC (Fels et al., 2016 Pethő et al., 2019 Sharma et al., 2019). The functional implications of this mechanosensitive expression have not yet been published. The decreased TRPV4 mRNA expression upon mechanical stimulation can be explained as a compensatory response of the cells which prevents Ca 2+ overload following the pressure stimulus (Fels et al., 2016).

Notably, PSCs also release TGF-β upon stimulation with pressure (Sakata et al., 2004 Fels et al., 2016). TRPV4 integrates mechanical stimuli and soluble signals such as TGF-β, and it drives the epithelial–mesenchymal transition (EMT) (Adapala et al., 2013 Sharma et al., 2019). TRPV4 expression is dramatically increased in many tissue samples of patients with liver fibrosis (Songa et al., 2014). Furthermore, TRPV4 is highly expressed in hepatic stellate cells (Songa et al., 2014). Inhibition of TRPV4 decreases cell proliferation of hepatic stellate cells, decreases their TGF-β�pendent activation and the expression of collagen 㬑 and α-smooth muscle actin genes in in vitro cultures (Songa et al., 2014). Inhibition of TRPV4 also leads to an increase in apoptosis and inhibition of autophagy in the TGF-β–treated hepatic stellate cell line HCS-T6. These findings can be taken as indication for a similar role of TRPV4 channels in PSCs as well.

Recording and Analysis of Intracellular Electrophysiological Signals


Ionic Channels of Excitable Membranes, 2nd edn, by Bertil Hille. Sinauer Associates (1992) . An excellent introduction to ion channels and the underlying theory. Required reading for the intracellular electrophysiologist.

Single-Channel Recording, 2nd edn, Bert Sakmann & Erwin Neher (eds). Plenum Pub. Corp. (1995). The definitive text on patch clamping principles and practice, by many of the originators of the technique.

Microelectrode Techniques: The Plymouth Workshop Handbook, 2nd edn, David Ogden (ed.). Company of Biologists, Cambridge (1994). A practical introduction to electrophysiological techniques. The course book for the annual microelectrode techniques course held at the Plymouth Marine Laboratory in the UK.

The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques, Rivka Sherman-Gold (ed.). Axon Instruments Inc. (1993). A useful (although pre-Windows) guide to electrophysiological data acquisition techniques, produced by one of the leading commercial suppliers in this field.


Angelo, K. & Margrie, T. W. Sci. Rep. 1, 50 (2011).

Padmanabhan, K. & Urban, N. N. Nature Neurosci. 13, 1276–1282 (2010).

Marsat, G. & Maler, L. J. Neurophysiol. 104, 2543–2555 (2010).

Angelo, K. et al. Nature 488, 375–378 (2012).

Stocks, N. G. Phys. Rev. Lett. 84, 2310–2313 (2000).

Longtin, A. & Mejias, J. F. Phys. Rev. Lett. 108, 228102 (2012).


In this review, we have highlighted the effects of ES as a physical stimulator on cellular behavior for the purposes of applying to regenerative medicine and tissue engineering. In most cases, ES facilitates cell proliferation and differentiation, enhance cell cathode migration and alignment to field vectors, and mainly through EGFR, PI3K and Ca 2+ related mechanism. ES can be delivered through tissue engineering scaffolds made of metallic biomaterials, conducting polymers, or carbon materials, the main methods are direct coupling, capacitive coupling, and using an electromagnetic field.

In the future, the combination of specific material/structure and ES will offer many advantages over other types of stimulations and allow for precise cellular regulation. Developing safe and effective partition-type scaffold combine with ES that can distinguish different areas to perform different stimulation still requires some challenges to be overcome. Although still in its early stages, the field of ES is rapidly evolving, and new next-generation regenerative medicine and tissue engineering will make it possible to take advantage of ES. Studying the low risk ES method for wearable application is also a future direction. With increasing research, electrical stimuli have the potential to play a significant role in tissue engineering and regenerative medicine.


How do 5 basic tastes turn into the myriad complex taste sensations we experience when eating food? Olfaction plays an important role in the perception of flavor, as do vision and touch. Taste information combines with information from these other sensory systems in the orbitofrontal cortex located in the frontal lobe. This region is believed to be important for the pleasant and rewarding aspects of food. Additionally, as taste is processed in higher-order regions of the CNS, information is combined using population coding mechanisms. Test how your senses combine to create flavor at home!

  • Taste cells express specific taste receptors and are located in taste buds within the papillae
  • Salt and sour taste cells rely on ion channels to depolarize the cell and release serotonin
  • Bitter, sweet, and umami taste cells rely on G-protein coupled receptors and second messengers that open ATP channels
  • At the level of the taste receptor cells, taste is perceived by using labeled line coding
  • Multiple regions in the mouth and throat play a role in processing of taste
  • Three cranial nerves innervate the tongue and throat
  • The cranial nerves synapse in the nucleus of the solitary tract in the medulla. Information then travels to the ventral posterior medial nucleus of the thalamus and then to the gustatory cortex
  • To perceive complex flavors, information from other sensory systems is combined with taste information in the orbitofrontal cortex

Watch the video: Water Changes Everything. (December 2021).