Calcium permeation and blocking effect in human leukaemia cells
Abstract
Cell-attached and inside-out patch-clamp methods were employed to identify and characterize mechanosensitive (MS) ionic channels in the plasma membrane of human myeloid leukaemia K562 cells. A reversible activation of gadolinium-blockable mechanogated currents in response to negative pressure application was found in 58 % of stable patches (n = 317). I-V relationships measured with a sodium-containing pipette solution showed slight inward rectification. Data analysis revealed the presence of two different populations of channels that were distinguishable by their conductance properties (17.2 ± 0.3 pS and 24.5 ± 0.5 pS), but were indistinguishable with regard to their selective and pharmacological properties. Ion-substitution experiments indicated that MS channels in leukaemia cells were permeable to cations but not to anions and do not discriminate between Na+ and K+. The channels were fully impermeable to large organic cations such as Tris+ and N-methyl-d-glucamine ions (NMDG+). Ca2+ permeation and blockade of MS channels were examined using pipettes containing different concentrations of Ca2+. In the presence of 2 mm CaCl2, when other cations were impermeant, both outward and inward single-channel currents were observed; the I-V relationship showed a unitary conductance of 7.7 ± 1.0 pS. The relative permeability value, PCa/PK, was equal to 0.75, as estimated at physiological Ca2+ concentrations. Partial or full inhibition of inward Ca2+ currents through MS channels was observed at higher concentrations of external Ca2+ (10 or 20 mm). No MS channels were activated when using a pipette containing 90 mm CaCl2. Monovalent mechanogated currents were not significantly affected by extracellular Ca2+ at concentrations within the physiological range (0-2 mm), and at some higher Ca2+ concentrations.
References
2. Kubohara, Y. and Hosaka, K. (1999). The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells. Biochemical and Biophysical Research Communications.;263:790–796.
3. Sachs, F. and Morris, C. E. (1998). Mechanosensitive ion channels in nonspecialized cells. Reviews of Physiology Biochemistry and Pharmacology.;132:1–77.
4. Vanoye, C. G. and Reuss, L. (1999). Stretch-activated single K+ channels account for whole-cell currents elicited by swelling. Proceedings of the National Academy of Sciences of the USA.;96:6511–6516.
5. Levitan, I. and Garber, S. S. (2000). Voltage-dependent inactivation of volume-regulated Cl− current in human T84 colonic and B-cell myeloma cell lines. Pflügers Archiv.
6. Levitan, I. Almonte, C. Mollard, P. and Garber, S. S. (1995). Modulation of a volume-regulated chloride current by F-actin. Journal of Membrane Biology.;147:283–294.
7. Martin, D. K. Bootcov, M. R. Campbell, T. J. French, P. W. and Breit, S. N. (1995). Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines. Journal of Membrane Biology.;147:305–315.
8. Egee, S. Mignen, O. Harvey, B. J. and Thomas, S. (1998). Chloride and non-selective cation channels in unstimulated trout red blood cells. Journal of Physiology.;511:213–224.
9. Negulyaev, Y. A. Vedernikova, E. A. and Maximov, A. V. (1996). Disruption of actin filaments increases the activity of sodium-conducting channels in human myeloid leukemia cells. Molecular Biology of the Cell.;12:1857–1864.
10. Negulyaev, Y. Khaitlina, S. Hinssen, H. Shumilina, E. and Vedernikova, E. (2000). Sodium channel activity in leukemia cells is directly controlled by actin polymerization. Journal of Biological Chemistry.;275:40933–40937.
11. Starushchenko, A. Mamin, A. Neguliaev, I. and Vedernikova, E. A. (2000). Activation of mechanosensitive ion channels in the plasma membrane of K562 cells. (in Russian). Tsitologiia.;42:669–674.
12. Hamill, O. Marty, A. Neher, E. Sakmann, B. and Sigworth, F. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv.;391:85–100.
13. Hille, B. (1992). Ionic Channels in Excitable Membranes. Sunderland, MA, USA: Sinauer Associates.
14. Negulyaev, Y. A. Maximov, A. Vedernikova, A. and Katina, E. (1997). Voltage-insensitive Na channels of different selectivity in human leukemic cells. General Physiology and Biophysics.;16:163–173.
15. Colquhoun, D. and Sigworth, F. J. (1995). Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E. , editors. Single Channel Recording. 2. New York: Plenum;. pp. 483–587.
16. Hamill, O. P. (1983). Potassium and chloride channels in red blood cells. In: Sakmann B, Neher E. , editors. Single Channel Recording. 2. New York: Plenum;. pp. 451–471.
17. Maximov, A. Vedernikova, A. Hinssen, H. Khaitlina, S. and Negulyaev, A. (1997). Ca-dependent regulation of Na+-selective channels via actin cytoskeleton modification in leukemia cells. FEBS Letters.;412:94–96.
18. Hamill, P. and McBride, D. W. (1997). Induced membrane hypo/hyper-mechanosensitivity: a limitation of patch-clamp recording. Annual Review of Physiology.;59:621–631.
19. Asahi, K. Sakurai, A. Takahashi, N. Kubohara, Y. Okamoto, K. and Tanaka, Y. (1995). DIF-1, morphogen of Dictyostelium discoideum, induces the erythroid differentiation in murine and human leukemia cells. Biochemical and Biophysical Research Communications.;208:1036–1039.
20. Kubohara, Y. (1999). Effects of differentiation-inducing factors of Dictyostelium discoideum on human leukemia K562 cells: DIF-3 is the most potent anti-leukemic agent. European Journal of Pharmacology.;381:57–62.
21. Christensen, O. (1987). Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels. Nature.;330:66–68.
22. Lumpkin, E. A. and Hudspeth, A. J. (1995). Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells. Proceedings of the National Academy of Sciences of the USA.;92:10297–10301.
23. Hoyer, J. Kohler, R. Haase, W. and Distler, A. (1996). Up-regulation of pressure-activated Ca2+-permeable cation channel in intact vascular endothelium of hypertensive rats. Proceedings of the National Academy of Sciences of the USA.;93:11253–11258.
24. Yeh, T. H. Herman, P. Tsai, M. C. Tran Ba Huy, P. and Van Den Abbeele, T. (1998). A cationic nonselective stretch-activated channel in the Reissner's membrane of the guinea pig cochlea. American Journal of Physiology.;274:C566–C576.
25. Kizer, N. Guo, X. L. and Hruska, K. (1997). Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proceedings of the National Academy of Sciences of the USA.;94:1013–1018.
26. Guharay, F. and Sachs, F. (1984). Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. Journal of Physiology.;352:685–701.
27. Achard, M. Bubien, K. Benos, J. and Warnock, D. G. (1996). Stretch modulates amiloride sensitivity and cation selectivity of sodium channels in human B lymphocytes. American Journal of Physiology.;270:C224–C234.
28. Benos, D. Awayda, S. Ismailov, I. and Johnson, P. (1995). Structure and function of amiloride-sensitive Na+ channels. Journal of Membrane Biology.;143:1–18.
29. Negulyaev, Y. Vedernikova, E. and Mozhayeva, N. (1994). Several types of sodium-conducting channel in human carcinoma A-431 cells. Biochimica et Biophysica Acta.;1194:171–175.
30. Taglietti, V. and Toselli, M. (1988). A study of stretch-activated channels in the membrane of frog oocytes: interactions with Ca2+ ions. Journal of Physiology.;407:311–328.
31. Bubien, K. and Warnock, G. (1993). Amiloride-sensitive sodium conductance in human B lymphoid cells. American Journal of Physiology.;265:C1175–C1183.
32. Yang, C. and Sachs, F. (1989). Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science.;243:1068–1071.
33. Zhang, Y. and Hamill, P. (2000). Calcium-, voltage- and osmotic stress-sensitive currents in Xenopus oocytes and their relationship to single mechanically gated channels. Journal of Physiology.;523:83–99.
34. Bradford, A. Ismailov, I. Achard, M. Warnock, D. Bubien, K. and Benos, D. (1995). Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes. American Journal of Physiology.;269:C601–C611.
35. Corey, D. and Garcia-Anoveros, J. (1996). Mechanosensation and the DEG/ENaC ion channels. Science.;273:323–324.
