A Review on Ion Channels and Gap Junctions: a Comparative

International Journal of Research ISSN NO:2236-6124

A Review on Ion Channels and Gap Junctions: A Comparative

Account on Normal Cell Proliferation and Cancer Progression

Ayyagari Ramlal1 and Laxman Kumar2*

1Department of Botany, Affiliated to Hindu College, University of Delhi, India

2Department of Botany, Jamia Hamdard, New Delhi, India

Abstract Ion channels are accountable for numerous extracellular and intracellular physiological processes where they contribute to maintaining ion homeostasis, signalling and potential of the membrane. Cancer, one amongst deadliest among all diseases, is outlined as a group of diseases caused by the unrestricted proliferation of normal cells that overlooks the natural laws of cell division. Ion channels have a pivotal role in cell proliferation. Involvement of ion channels in the progression of cancer has been revealed long back and deciphering the underlying principles involved in the ion channel-mediated cancer progression has been a major sphere in recent research. The cell interactions are essential to multicellular organisms for their development and required for the normal functioning of the body which includes tight and gap junctions. Gap junctions are intracellular channels allowing to and fro movements of ions across cells. This article emphasizes the potentialities of some voltage-gated ion channels (VGICs) including Chloride, Potassium and Sodium (VGKCs, VGSCs and CLCAs) and gap junctions which have been identified during cancer development and along with some therapeutic controls. The expression of these channels in some cases is directly concerned with the cancer development whereas some others show indirect participation. Ion channels have two roles in the promotion of abnormality within cells and regulation or forestalling of the signalling cascade. Cell-to-cell interactions provided by Gap junctions (GJs) could lead to cause serious complications like cancer once they go unregulated and altered.

Keywords: Voltage-gated ion channels (VGICs), ion channels, cancer progression, gap junctions and therapeutic controls

1. Introduction

Involvement of ion channels in normal cell division

Ions have played significant roles in signalling pathways across the cell membrane and other membranes. Monovalent ions like Sodium and Potassium primarily play a job in the adjustment of membrane potential whereas the divalent ions like Magnesium, Zinc and Calcium are crucial for intracellular signalling processes and act as secondary messengers. [1] Fundamental processes like contraction of muscles, secretion, the synaptic transmission including generation

*Corresponding author: [email protected] | +91 9911425929

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and propagation of action potential is all mediated by ion channels. [3] Expression of various ion channels in cells during the adaptive and innate immunity and their opening control the influx-efflux of ions via the plasma membrane (PM) and endoplasmic reticulum or lysosomes or mitochondrion. [1] Cell division and cell proliferation is a very complex phenomenon is under the tight regulation of proteins, molecules and various ions. ATP, calcium ions, cyclin-dependent kinases (CDKs) and cyclins all are part of the cell cycle machinery. The chief component which maintains the cell cycle is its membrane potential. The electrical potential is generated by the ion transporters and channels which is created by the intra- and extracellular ion concentration. There was less or no mitoses in the cells like muscle and neuron which showed much-hyperpolarised resting potential. The excitable roles of ion channels include generation of action potentials and contraction in neurons and muscles respectively. Apart from these functions, voltage-gated channels also include in non-excitable roles like homeostasis maintenance via regulating ion transport and volume regulation. [2] Cells are continuously degraded and recycled by the process of autophagy, studies revealed that ion fluxes created across membranes have played a major role in the regulation of autophagy. This process supports other physiological processes as well like cell differentiation, embryonic development and immunity and its malfunction may cause cardiovascular, infections, metabolic diseases and cancer as well. [3] Cell cycle is under the tight regulation of voltage-gated potassium channels, experiments carried on unfertilised oocytes of mouse showed that activity remained high during the M and G1 phase, while it reduced during synthesis (S) and G2 phases. [19] Gap junctions represent the group of internal membrane proteins that direct the exchange of amino acids, small peptides, ions and sugars across the neighbouring cells. Connexins form the gap junctions in vertebrates while in invertebrates it is formed from innexins. Cx43, Cx40 and Cx45, the dominant isoforms of connexins-gap junctions show their expression in the heart and play a major role in carrying the currents between the myocardium cells. These GJs are unevenly distributed in the ventricle and atrial regions thereby resulting in anisotropy of heart tissues. [21]

The transition from normal to abnormal cell proliferation

Ion channels are made up of macromolecular proteins that span through the cell membrane via lipid bilayer and allows the passage of ions and are essential for cell proliferation along within the development of cancer as well [4, 5]. As a result of protein structural conformational changes, it causes the channel to open. Small changes happened in the structure allows the in and out the movement of ions up to 10 million ions/sec. It consists of an aqueous pore which is accessible to selective ions. Depending upon the opening agent, they can be broadly divided into voltage-gated and ligand-gated, the former one requires the potential difference across the membranes to open while the latter one requires a particular signal which can activate their channelling like neurotransmitters channels [4]. Cancer is marked by the abnormal growth and proliferation of cells which encompasses the alteration of genes and/or proteins which are required for fundamental processes of survival, proliferation, differentiation and cell death [7]. It is classified into many categories based on the site of production of an abnormal mass of cells. The channels and carriers which transport ions across the membranes have a vital role in the regulation of cell survival, death and mortality. The altered regulation of these channels may result in the transformation of normal cells into cancerous cells. Many studies show that alterations or modifications in the channels or gates may impact various cascades related to cancer growth like tumour cell survival and proliferation, malignant progression and invasive behaviour of normal cells. (Florian Lang, 2014) [6]. Calcium-mediated

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channels, K+ channels, Sodium and other anions channels have shown their roles in the enhancement of tumour and its metastasis. The present concern among the research community is that the natural process of cell death is not followed by the cancerous cells, therefore the resistance and insensitivity acquired by cancerous cells against apoptosis must be overcome. [13] The transmuted expression of a channel can be utilised for various diagnostic purposes; recent shreds of evidence show that blocking the activity of channels results in impediment of tumour growth. The transient receptor potential protein (TRPs) act as regulators of stimuli like temperature, vision, taste and other sensory responses via ion fluxes. A subfamily of TRPs called TRPM (TRP-Melastatin) which function in regulating the cationic fluxes such as K+, Ca2+, and Mg2+ etc. [15] Expression anomalies of connexions have known to cause diseases like cardiac hypertrophy and ischemia. Recent studies revealed that gap junctions implicated in Charcot-Marie-Tooth and visceroatrial heterotaxia syndrome (CMT and VHS) inherited diseases, [22] It was observed that increased expression levels of mRNA and protein were observed in some tumours along with mislocalization of connexins, for instance, prostate cancer has Cx43 and Cx32 and pancreatic and colon cancer showed Cx26 expression. [23]

Cationic channels

1. Potassium channels

Studies revealed that overexpression of calcium-activated K+ channel called BK channels one of the voltage-gated ion channels are entailed in the progression of cancer. Huge genetic diversity and channel properties within potassium channels can be seen (V. Suppiramaniam et al, 2017). These BK channels are found in a broad range of cells and are energised by the increasing levels of intracellular calcium and other molecules and mechanisms. [7] They are crucial for the release of neurotransmitters, muscle relaxation along with processes involved in tumorigenesis. In vitro studies revealed that glioma cell growth, survival and migration are strongly controlled by BK channels. Mansoor Abdul et al (2003) showed that potassium channels also participate in the proliferation of breast cancer. The T-lymphocytes are dependent on the voltage-gated K channels (VGPCs) for their activation, kv1.3 is an essential component which activates the peripheral T-cells. [8] the drugs like dequalinium and amiodarone are potent growth suppressors as they bind to the K-channels and inactivate them thus, control the proliferation of MCF, a cell line of breast cancer. Kv1.1 overexpression is seen in medulloblastoma (Alisa Litan et al., 2015 and Northcott et al., 2012), increased levels are seen in the cancers like breast, colon and prostate. Constitutive expression of Kv11.1 is seen in neuroblastoma. [11] Also VGPCs like, KCa3.1 and Kv10.1 are involved in breast cancer, studies carried out by the group of H. Ouadid-Ahidouch (Amiens, France), used in controlling cell proliferation in melanoma cells using these K+ channels (a group led by R. Schonherr, Jena, Germany). Another VGPCs called Kv11.1 is proved to be a good therapeutic target as it accelerates apoptosis when complexed with β1 integrin in the acute lymphoblastic leukaemia (AML). [12] Roles of K+ and Cl- channels have been reported in the primary brain tumours and gliomas where they promote the invasiveness and formation of brain metastasis. [14] Potassium channel family-like, ether à go-go (EAG) are nowadays gaining popularity as this tool can be used to detect and in the therapy of various cancers. [16] Pieces of evidence provided by the Patch-clamp technique showed that potassium channel changes as cancer propagates. [17] Many studies revealed that impaired or malfunctioned expression of K- channels leads to cause cancer, like high Kv11.1 levels of expression are responsible for both blood and solid cancer while Kv10.1 expression is seen in over 70% of organ-linked

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cancer types in humans. [18] Glioblastoma, one of the fatal cancer forms affecting the brain and are dependent on calcium signalling. As the concentration of calcium increases in the cells of glioblastoma, it activates the Phospholipase C/Inositol -1,4,5-triphosphate (PLC/IP3) pathway. The cascade thereby activates further pathways among one is in the 2+ increase of cytosolic Ca which further activates the KCa3.1 channel, this suggests that 2+ KCa3.1 channel are involved modifications of Ca oscillations leading in the migration process of glioblastoma cells. [20] Potassium channels act as probable targets for anticancer therapies therefore, K+ channel antibodies and blockers are used. A drug like clofazimine acts by inhibiting the Kv3.1 in chronic lymphocytic leukaemia thereby promoting neoplastic B cell death. [24] Blockers of Kv11.1 extracted from scorpion venom (KAaH1 and KAaH2) can be used in the cancers like adenocarcinoma, breast and glioblastoma by inhibiting the cell migration and adhesion without effecting the apoptosis and cell cycle.[24] Table 1 represents various potassium channels and their involvement in various cancer forms.

Table 1. Sodium Channel and its Subtype Expression in Cancer

S No Cancer type Channel gene References 1 Breast SCN5A (Nav1.5) 30 SCN8A (Nav1.8) SCN9A (Nav1.9) SCN1B (β1) SCN2B (β2) SCN4B (β4) 2 Cervix SCN2A (Nav1.2) 30 SCN4A (Nav1.4) SCN8A (Nav1.8) SCN9A (Nav1.9) SCN1B (β1) SCN2B (β2) SCN4B (β4) 3 Colon SCN5A (Nav1.5) 30 4 Lung Small cell 30 SCN3A (Nav1.3) SCN5A (Nav1.5) SCN8A (Nav1.8) SCN9A (Nav1.9) Non-small cell SCN5A (Nav1.5)

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SCN8A (Nav1.8) SCN9A (Nav1.9) SCN1B (β1) SCN2B (β2) SCN3B (β3) SCN4B (β4) 5 Lymphoma SCN5A (Nav1.5) SCN8A (Nav1.8) SCN9A (Nav1.9) SCN11A (Nav1.11) 6 Melanoma SCN8A (Nav1.8) 30 7 Mesothelioma SCN2A (Nav1.2) SCN8A (Nav1.8) SCN9A (Nav1.9) 8 Neuroblastoma SCN5A (Nav1.5) 30 9 Ovary SCN1A (Nav1.1) 30 SCN2A (Nav1.2) SCN3A (Nav1.3) SCN4A (Nav1.4) SCN5A (Nav1.5) SCN9A (Nav1.9) 10 Prostate SCN2A (Nav1.2) 30 SCN3A (Nav1.3) SCN4A (Nav1.4) SCN8A (Nav1.8) SCN9A (Nav1.9) SCN1B (β1) SCN2B (β2) SCN3B (β3) SCN4B (β4)

A – α subunit and β – β subunit (modified from William J. Brackenbury, 2012)

2. Role of Sodium ion channels in the development of cancer

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When patients suffering from gastric cancer (GC) were assessed, they tested positive for the presence of VGSCs (Voltage-gated Sodium Channels). The study involved the expression of gene SCN9A which encodes the VGSC called Nav1.7, which is expressed in abundance during the GC. Prognosis of GC was evaluated by the immunohistochemical analysis which uses transporter NHE1 (Na+/H+ exchanger -1) and an oncoprotein metastasis-associated colon cancer 1 (MACC1). The results yielded that voltage-gated sodium currents were reduced the Nav1.7 was suppressed during the same time, expression of NHE1 was decreased and the increased extracellular pH was and intracellular pH was decreased. These changes led to the invasion suppression and proliferation of GC cells in the mice. Therefore, it can be inferred that NHE1 was upregulated by MACC1, it increases Nav1.7 channel which promotes the gastric cancer progression. [9] Further, NHE1 if also found to be involved in breast cancer and melanoma metastasis. [11] Evidence presented by J.-Y. Le Guennec (Tours, France) showed that the activity of Nav1.5, a VGSC, increased during the proliferation rate of breast cancer cells in humans mediated by increased activity in cysteine cathepsin. The ion channel expression in smooth muscle cells was studied using repressor element -1 silencing Transcription factor (REST) and revealed that it regulates Nav1.2, which is involved in cancer development along with other diseases. The downregulation of REST, upregulates the KCa3.1, this further lead in the membrane hyperpolarisation and movement of calcium ions. [12] The activation of VGSCs gets triggered by membrane depolarisation and in turn which generates sodium currents creating action potential in muscle cells and neurons. Hodgkin and Huxley (1952), discovered the Sodium currents with the help of the voltage-clamp technique. [29] Nav are involved in regulating the proliferation and invasiveness of cells. There are 9 genes which encode different NaVα from NaV1.1 to NaV1.9 (SCN1A to SCN10A and SCN11A). these are composed of 4 homologous domains I -IV where each domain consists of 6 alpha-helical transmembrane spanning regions from S1 to S6. S1-S4 represent to constitute voltage-sensing domain (VSD) while remaining two form pores of the channel, separated by extracellular P loop. [29] There is a pore-blocking inhibitor called tetrodotoxin (TTX), which selectively inhibits NaV1.1-1.4, NaV1.6, and NaV1.7 and resistant towards NaV1.5 NaV1.8 and NaV1.9. Table 2 represents various sodium channels and their involvement in various cancer forms.

Table 2. Potassium channel expression in cancer

S No Cancer type Channel gene References

1 Adrenal Kir3.4 (KCNJ5) 18 K2P3.1 (KCNK3)

2 Bladder Kv1.5 24

3 Blood Kv1.5 (KCNA5) 18

Kv10.1 (KCNH1)

Kv11.1 (KCNH2)

4 Bone Kv10.1 (KCNH1) 18

KCa1.1 (KCNMA)

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5 Brain Kv1.5 (KCNA5) 18

Kv1.1 (KCNA1)

Kv10.2 (KCNH5)

Kv11.1 (KCNH2)

KCa1.1 (KCNMA)

KCa3.1 (IK1)

Kir4.1 (KCNJ10)

Kir6.1 (KCNJ8)

Kir6.2 (KCNJ11)

6 Breast Kv1.3 (KCNA3) 18

Kv4.1 (KCND1)

Kv10.1 (KCNH1)

Kv11.1 (KCNH2)

KCa1.1 (KCNMA)

KCa2.3 (SK3)

KCa3.1 (IK1)

Kir2.2 (KCNJ12)

Kir3.1 (KCNJ3)

Kir6.1 (KCNJ8)

Kir6.2 (KCNJ11) K2P5.1 (KCNK5) K2P9.1 (KCNK9)

7 Cervix Kv10.1 (KCNH1) 18

8 Endometrium Kv11.1 24

9 GI tract Kv4.1 (KCND1) 18

Kv7.1 (KCNQ1)

Kv10.1 (KCNH1)

Kv11.1 (KCNH2)

KCa2.3 (SK3)

KCa3.1 (IK1)

Kir2.2 (KCNJ12) K2P9.1 (KCNK9)

10 Head and neck Kv3.4 (KCNC4) 18

Kv10.1 (KCNH1)

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Kv11.1 (KCNH2)

11 Oral Head and neck Kv3.4 24

12 Kidney Kv10.1 (KCNH1) 18

Kv11.1 (KCNH2)

13 Lung Kv1.3 (KCNA3) 18

Kv11.1 (KCNH2)

Kir3.1 (KCNJ3) K2P9.1 (KCNK9)

14 Lymphoma Kv1.3 (KCNA3) 18

15 Melanoma Kv11.1 (KCNH2) 18

KCa2.3 (SK3)

KCa3.1 (IK1)

Kir6.1 (KCNJ8)

Kir6.2 (KCNJ11) K2P9.1 (KCNK9)

16 Ovary Kv10.1 (KCNH1) 18

Kv11.1 (KCNH2)

KCa1.1 (KCNMA)

17 Pancreas Kv1.3 (KCNA3) 18

Kv10.1 (KCNH1)

Kir3.1 (KCNJ3)

18 Parathyroid Kv1.3 24

19 Prostate Kv1.3 (KCNA3) 18

KCa1.1 (KCNMA)

KCa3.1 (IK1)

Kir2.2 (KCNJ12)

20 Sarcoma Kv10.1 (KCNH1) 18

21 Skeletal muscles Kv1.3 24

Kv1.5

22 Skin Kv1.3 24

Kv1.5

23 Stomach Kv1.4 24

Kv1.5

Kv4.1

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Kv10.1

Kv11.1

Kv2.1

24 Thyroid Kv11.1 (KCNH2) 18

25 Uterine Kir6.1 (KCNJ8) 18

Kir6.2 (KCNJ11)

Legend – positive for tumours with overexpression, the expression is inversely related with tumour and recurring somatic mutations, up-regulated and down- regulated (modified from Xi Huang et al, 2014 and Clara Serrano-Novillo et al, 2019)

Cancer and Calcium channels

Calcium is a signalling molecule, it is required for major metabolic processes like motility, apoptosis, contraction, in the release of transmitters, in endocytosis and exocytosis. These VG calcium channels (CCs) have been studied widely in excitable cells of cardiovascular and neuromuscular physiology and neuroscience while their inhibition is important to treat epilepsy and hypertension. [31] This family of ion channels comprise of 10 genes having sensor site for voltage along with binding sites for inhibitors and modulators. L-type Calcium channels are present in skeletal muscle, smooth muscle and bones having subunits are CaV1.1, CaV1.2, CaV1.3 and CaV1.4. The channel blockers of L- type calcium are used in the pharmacological field to treat hypertension by inhibiting the action potential of smooth muscle cells. P-type calcium channels (subunit is CaV2.1) are present in Purkinje neurons in the cerebellum. N-type calcium channels are present in the brain and peripheral nervous system. CaV2.2 is the N-type subunit. R-type, these type of calcium channels are present in the neurons and neuroblastoma cell [31] and CaV2.3 as its subunit. T-type calcium channel can be seen in thalamus, bone and neurons. CaV3.1, CaV3.2 and CaV3.3. Blockers of this ion channel reduce the neuronal calcium conductance and reduce the chance of epilepsy attack.

Anionic channels

1. Chloride channels and cancer

Chloride ion channels are ubiquitous and are present on organelles and plasma membrane where they regulate pH, ion homeostasis, electrical excitability and regulate cell volume. [10] Human Ca2+- activated chloride channels (CLCA2) were not expressed in breast tumours progression whereas they were synthesised normally in non- tumorigenic cell lines (Achim D. Gruber, 1999). p53, a tumour protein is a key master regulator in the arresting cell cycle and cell death thereby prevents the development of tumours. Now, when CLCA2 was expressed abnormally it results in curbing cancer cell migration. But siRNA- silenced CLCA2 gene stimulated the progression of cancer cells.

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This also resulted in the enhanced expression of FAK (Focal Adhesion Kinase), a regulator of cancer progression. It was concluded that p53 regulates the cancer cell migration and invasion by mediating with downstream CLCA2 and emphasised that is a direct target of p53 (Marta Peretti et al, 2014 and Yasushi Sasaki and colleagues, 2012). The intracellular chloride channels are also involved in the development of cancer, for instance, CLIC1 is a cytosolic protein but in the cases of tumorigenic growth and pathogenic conditions like neurodegenerative diseases, it translocates to the plasma membrane. CIC-3, a member of chloride channels, expression was seen in the androgenic process (prostate cancer) [10] also volume-activated chloride current density was higher in human nasopharyngeal carcinoma as compared to normal cell lines (Mao and colleagues, 2004). In HeLa cells, inhibition of CIC-3 protein expression results in the cessation of the cell cycle in S-phase, therefore proving that voltage-activated chloride channels are crucial in cell cycle-dependent migration of HeLa cells. [10]

Gap Junctions

Structure

Freeze fractured images show a dense array of particles forming mirror-image on the PM of adjacent cells (Chalcroft and Bullivant, 1970). The fundamental unit of a gap junction is a connexin; each connexin is composed of 6 polypeptides of connexins following oligomerisation and forms an aqueous pore which spans the PM at a single location. For the formation of a complete junction, 2 connexins from adjacent cells arrange themselves in such a manner that they form a continuous channel with their cytoplasm linked together. The pore size measures up to a diameter ranging from 0.8 to 1.4 nm allowing transport up to 1200 Da [25] The various characteristic features of GJs include; membrane-spanning channels must be present between adjacent cells and it should be selectively permeable and it should sink with proper timing and cell activity. [26]

Mechanism of action

Studies related to tumour promoters like 12-O-tetra-decanoylphorbol-13-acetate (TPA) showed that they are potent inhibitors of gap junctions thereby disrupting intracellular communication. When TPA was applied to mouse interfollicular skin cells, there was a significant reduction in gap junctions. Similar results were obtained when the avian sarcoma virus was used as an agent for study. [23] When Cx43 and GJA1 genes encoding proteins when mutated, they are known to cause frequent tumours like stomach adenocarcinoma and tumour progression may also have resulted from abnormal intracellular communication of GJ. Further, when metabolites and hormones are not transported across cells, due to impaired GJIC, this may lead to cause uncontrolled division of cells. [23, 27] Regular and continuous intracellular communications are necessary and essential for maintaining normal growth rate, this condition may escape in case of cancer development as sometimes tumour cells form their communicating channel for their progression. This abnormality can be occurred due to 1) due to the closure of membrane connexons 2) incompatible and altered expression of connexons and 3) reduced cell adhesion. Studies revealed that in liver carcinoma, the expression of Cx26 and Cx32 decreased permanently. Sometimes the tumour cells change the pattern of connexins during cancer ontology. [25] Sometimes the expression levels of connexins could be deceiving as in the case, Cx43 acts as tumour suppressor while Cx26 is seen during tumour growth. Thus, connexins assessment is not a better diagnostic tool to

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measure cancer progression. Studies carried with Cx43 and Cx32 revealed that they act as good tumour-suppressing agents by controlling rat glioma and hepatoma cells of human respectively. [23] Immunoelectron microscopic technique revealed a smaller number of GJs were identified in the basal cell carcinoma and squamous cell carcinoma when compared to normal skin. [28] Similarly, a smaller number of gap junctions were seen in the case of human laryngeal carcinoma when compared with normal epithelia of the larynx. [28] Table 3 gives the plausible roles of gap junction proteins – connexins expression as seen in rodents while table 4 shows the link between cancer and connexin in genetically modified mouse models.

Table 3. The table depicts the relationship between Greek letter and molecular mass nomenclature of gap junction proteins – connexins expression observed in Rodents

S No Molecular Greek letter Site of Remarks References Mass nomenclature expression [15] nomenclature in the organ [14] 1 Cx43 α1 Heart Prostate, 22, 23 pancreatic, breast, head and neck, non- small-cell lung and colorectal – Act as a tumor suppressor 2 Cx38 α2 Embryo - 22 3 Cx46 α3 Lens - 22 4 Cx37 α4 Lung - 22 5 Cx40 α5 Lung - 22 6 Cx45 α6 Heart Reduced 22 expression in colon and colorectal

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7 Cx33 α7 Testis - 22 8 Cx50 α8 Lens - 22 9 Cx32 β1 Liver Prostate 22, 23 10 Cx26 β2 Liver The 22, 23 expression is seen in pancreatic, colorectal and colon 11 Cx31 β3 Skin - 22 12 Cx31.1 β4 Skin - 22 13 Cx30.3 β5 Skin - 22

(Nalin M. Kumar et al, 1996 and Trond Aasen, et al, 2016)

Table 4. The relation between cancer and connexin in genetically modified mouse models

S No Connexins Gene Remarks Reference(s) encoding 1 Cx43 GJA1 Increased breast metastasis and lung tumour 2 Cx32 GJB1 Increased spontaneous 23 liver tumours in males, liver and multiple tumours, lung and adrenal, pituitary and intestinal tumours

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3 Cx26 GJB2 Increased breast cancer

Conclusions

It can be concluded that pumps and ion channels play prime roles in cell proliferation, migration, apoptosis and differentiation along with its primary responsibilities of regulating membrane potential, electric signalling and ion homeostasis. [11] The imbalance expression of the potassium, chloride and sodium ion channels lead to cancer. Various techniques like immunohistochemical analysis using a particular antibody, the involvement of ion channels can be assessed. [8] Also the levels of RNA can be analysed using Northern hybridisation thus, monitoring the levels of production of different transcripts during tumorigenesis. It can be stated that in cancer, the abnormal expression of NaV was found to involved in the initial migration and invasiveness of cells during organ development also VGSCs have an important role in the progression of cancer leading to metastasis. [29, 30]

Acknowledgement

We thank Dr D K Mallick and Dr Aparna Nautiyal, Department of Botany, Deshbandhu College, University of Delhi, New Delhi, India for helpful advice and discussion.

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