The Involvement of Ion Channels in Cell Proliferation

The control of cell proliferation involves diverse signaling pathways, growth factors, and receptors. The involvement of ion channels in this process is supported by a wealth of experimental evidence with mechanisms not always well understood. However, the emerging roles ion channels play in such cellular processes are related to changes in Ca²⁺ signaling, stressing its importance in proliferation initiation and maintenance. The use of specific ion channel blockers and antibodies enables researchers to unravel the complexities of this issue. Below we highlight the use of Alomone Labs’ ion channel antibodies and modulators in some aspects related to this research field.

The control of cell proliferation involves diverse signaling pathways, growth factors, and receptors. The involvement of ion channels in this process is supported by a wealth of experimental evidence. Growth factors increase expression of K⁺ channels¹. Primary culture of bone marrow derived marcrophages is a unique non transformed model in which proliferation and activation can be studied separately². In this system, the presence of K_V1.3 and K_ir2.1 was shown using immunocytochemical electron microscopic detection using Anti-K_V1.3 (KCNA3) Antibody (#APC-002) and Anti-K_ir2.1 (KCNJ2) Antibody (#APC-026). The number and density of these channels was upregulated by treatment with M-CSF (the specific growth factor for this cell type) as shown in immunoblots using the above antibodies. This proliferation was inhibited by the K_V1.3 specific blocker, Margatoxin (#STM-325). A wide variety of mitogenic factors activate the Na⁺/H⁺ exchanger, and many factors stimulate Na-K-2Cl cotransport³. One expected consequence of the activation of these transport systems is an increase of cell volume. Expression and single channel activity of some ion channels has been shown to vary during the cell cycle⁴, most notably during the transition from G₀ or G₁ to S phase. Czarnecki et al.⁵ have shown that the peak density of the transient outward K⁺ current (Ito) expression in the GH3 pituary cell is upregulated in quiescent cells compared with proliferating cells. Increase in Ito expresssion is accompanied by an increase in K_V1.4 alpha subunit protein levels, a decrease in K_V1.5 protein levels and no change in K_V4.3 protein levels. This was shown by western blot using antibodies specific for each subunit: Anti-K_V1.4 Antibody (#APC-007), Anti-K_V1.5 (KCNA5) Antibody (#APC-004) and Anti-K_V4.3 Antibody (#APC-017). The functional contribution of K_V4.3 to the Ito current was confirmed using Phrixotoxin-2 (#STP-710), a specific blocker of K_V4.3 channels. As chloride channels are criticial to the cell cycle in vascular smooth muscle cells, the identification of the specific channel involved is of interest. Wang et al.⁶ used Anti-CLC-3 (CLCN3) Antibody (#ACL-001) to show expression of ClC3 in VSMCs and functional enhancement by endothelin-1.

Cell proliferation must, at some point, increase cell volume since all cell components must be duplicated prior to cell division. Changes in cell volume require participation of ion transport¹ and rearrangements of the cytoskelton⁷. Cell proliferation has been shown to correlate with increases in cell volume in fibroblasts⁸, mesangial cells⁹, lymphocytes¹⁰, HL-60 cells¹¹, GAP A3 hybridoma cells¹², smooth muscle cells¹³, and HeLa cells¹⁴. In human T-lymphocytes K_V1.3 is involved in volume regulation since blocking the channel with Agitoxin-2 (#STA-420) increased the maximal swelling of the cells¹⁵. Depolymerization of actin filaments leads to disinhibition of the Na⁺/H⁺ exchanger and/or Na-K-2Cl transporter, resulting in cell swelling¹⁶. Notably, increased channel activity is found in cancer cells¹⁷. Pillozzi et al¹⁸. used Anti-KCNH2 (HERG) Antibody (#APC-062) to detect K_V11.1 (HERG) channels in the leukemic cell line FLG-29.1 and in primary leukemic cells. They further showed that a specific K_V11.1 inhibitor caused a strong inhibition of colony formation when the cells were seeded in semisolid medium. The presence of K_V11.1 protein was also shown in four hematopoetic cell lines, CEM, K562, U937 and HL-60 by Smith et al.¹⁹, using Anti-KCNH2 (HERG) Antibody. They also showed that by blocking the K_V11.1 currentwith E-4031 (#E-500) significantly decreased the number of cells.

The precise mechanisms by which ion channels regulate proliferation are not understood. Two major hypotheses attempt to explain this mechanism: regulation of membrane potential and regulation of cell volume. Phosphorylation/dephosphorylation has also been implicated.²⁰ K⁺ channels maintain the membrane potential at negative values, providing a large driving force for calcium entry into cells. The elevated [Ca²⁺]_i (the Ca²⁺concentration generated by calcium influx) is required for the cell’s ability to proliferate.²¹ In Muller cells, the activity of calcium dependent postassium channels (BK) was connected to the mitogenic action of ATP. Blocking these channels using Charybdotoxin (#STC-325) and Iberiotoxin (#STI-400) fully inhibited the effect of ATP on DNA synethesis.²² Membrane potential also regulates transport of (sodium-coupled) nutrients, which also affects the cell’s ability to proliferate. According to the volume hypothesis,²³ K⁺ channel activity controls the ion influx-efflux ratio and cell volume. If secondary volume regulating mechanisms are not activated, K⁺channel blockade induces cell swelling. In turn, cell swelling dilutes the intracellular concentration of a solute controlling the expression or activity of genes or enzymes involved in DNA synthesis. As a corollary of this hypothesis, any change in cell volume should be associated with a change in cell proliferation. Whether the proliferative effect is a consequence of a change in ion channel number or a change in open probability, must be considered as well, although most studies have not explored this point.²⁴ In addition, the differences between excitable and nonexcitable cells must be examined; the control of proliferation in cells that can produce an action potential may not be the same as in other cells. The control of proliferation in neoplastic cells may be governed by ion channels not expressed in their normal counterparts.

It should be noted that although both membrane potential and cell volume are regulated by ion channels, their relationship need not be linear; cell swelling can be induced by agents that do not affect membrane potential.²⁵ In addition, cell proliferation and changes in membrane potential/cell volume occur on different time scales – days vs. hours or shorter, respectively.

The effect of specific channel blockers on cell proliferation is cell type-specific. Charybdotoxin neither modified membrane current at a holding potential of -70 mV, nor affected cell volume, so it is not surprising that it did not block proliferation of C6 glioma cells.²³ However, in T-cells, Charybdotoxin fully inhibited IK current²⁴ and blocked cell proliferation by 67%. In porcine granulosa cells, in contrast, inhibition of K_V1.3 with Charybdotoxin and Margatoxin caused an increase in cell proliferation when the cells were concomitantly treated with FSH (follicle stimulating hormone).²⁵ In these cells, the depolarization associated with Charybdotoxin and Margatoxin inhibition of K_V1.3 enhances calcium influx (in most cells, blockade of K⁺ channels decreases calcium influx), thus increasing cell proliferation. This seems to be dependent upon the type of Ca²⁺-conducting channels available in a particular cell, whether they are voltage-dependent or constitutive. Schwann cells have been shown to contain a variety of K⁺ channels by the use of specific antibodies: Anti-K_V1.1 (KCNA1) Antibody (#APC-009), Anti-K_V1.2 (KCNA2) Antibody (#APC-010), Anti-K_V1.4 Antibody, Anti-K_V1.5 (KCNA5) Antibody, Anti-K_V3.1b (KCNC1) Antibody (#APC-014) and Anti-K_V3.2 (KCNC2) Antibody (#APC-011). The expression of these channels was shown to be developmentally regulated. The specific blockers Charybdotoxin, Agitoxin-2 and Dendrotoxin-I (#D-390) did not affect cell proliferation, whereas non-specific blockers such as TEA, barium and clofilium did.²⁶

References

1. Lang, F. et al. (2005) J. Membr. Biol. 205, 147.
2. Vicente, R. et al. (2003) J. Biol. Chem. 278, 46307.
3. Ritter, M. et al. (1997) Eur. J. Cell. Biol. 72, 222.
4. Premack, B.A. and Gardner, P. (1991) J. Clin. Immunol. 11, 225.
5. Czarnecki, A. et al. (2003) Am. J. Physiol. Cell Physiol. 284, 1054.
6. Wang, G.L. et al. (2002) Circ. Res. 91, 28.
7. Cantiello, H.F. (1995) Kidney Int. 48, 970.
8. Lang, F. et al. (1992) Cell. Physiol. Biochem. 2, 213.
9. Yamamoto, T. et al. (1991) Kidney Int. 40, 705.
10. Dingley, A.J. et al. (1992) Biochemistry, 31, 9098.
11. Brennan, J.K. et al. (1991) J. Cell. Physiol. 146, 425.
12. Needham, D. (1991) Cell. Biophys. 18, 99.
13. Feltes, T.F. et al. (1993) Am. J. Physiol. 264, C169.
14. Takahashi, A. et al. (1993) Am. J. Physiol. 265, C328.
15. Khanna, R. et al. (1999) J. Biol. Chem. 274, 14838.
16. Lang, F. et al. (1998) Physiol. Rev. 78, 247.
17. Nilius, B. and Wohlrab, W. (1992) J. Physiol. 445, 537.
18. Pillozzi, S. et al. (2002) Leukemia, 16, 1791.
19. Smith, G.A. et al. (2002) J. Biol. Chem, 277, 18528.
20. Villaz, M. et al. (1995) J. Physiol. 488, 689.
21. Ullrich, N. (1999) Neuroscientist 5, 70.
22. Moll, M. et al. (2002) Invest. Ophthalmol. Vis. Sci. 43, 766.
23. Rozaire-Dubois, B. et al. (2000) Pflugers Arch. 440, 881.
24. Jensen, B.S. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10917.
25. Manikkam, M. et al. (2002) Biol. Reprod. 67, 88.
26. Sobko, A. et al. (1998) J. Neurosci. 18, 10398.