Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Oct;87(19):7579–7582. doi: 10.1073/pnas.87.19.7579

Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

C M Armstrong 1, C Miller 1
PMCID: PMC54791  PMID: 2217187

Abstract

Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K+ channels in an altered, debased conformation. Voltage-dependent K+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K+ channels. These expressed channels respond to low Ca2+ identically to voltage-dependent K+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltage-dependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K+ channels and is required to stabilize the native conformations of these membrane proteins.

Full text

PDF
7580

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong C. M., Lopez-Barneo J. External calcium ions are required for potassium channel gating in squid neurons. Science. 1987 May 8;236(4802):712–714. doi: 10.1126/science.2437654. [DOI] [PubMed] [Google Scholar]
  2. Armstrong C. M. Potassium pores of nerve and muscle membranes. Membranes. 1975;3:325–358. [PubMed] [Google Scholar]
  3. Baumann A., Grupe A., Ackermann A., Pongs O. Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems. EMBO J. 1988 Aug;7(8):2457–2463. doi: 10.1002/j.1460-2075.1988.tb03092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Butler A., Wei A. G., Baker K., Salkoff L. A family of putative potassium channel genes in Drosophila. Science. 1989 Feb 17;243(4893):943–947. doi: 10.1126/science.2493160. [DOI] [PubMed] [Google Scholar]
  5. Filimonov V. V., Pfeil W., Tsalkova T. N., Privalov P. L. Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin. Biophys Chem. 1978 May;8(2):117–122. doi: 10.1016/0301-4622(78)80003-9. [DOI] [PubMed] [Google Scholar]
  6. Frech G. C., VanDongen A. M., Schuster G., Brown A. M., Joho R. H. A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning. Nature. 1989 Aug 24;340(6235):642–645. doi: 10.1038/340642a0. [DOI] [PubMed] [Google Scholar]
  7. Gilbert D. L., Ehrenstein G. Effect of divalent cations on potassium conductance of squid axons: determination of surface charge. Biophys J. 1969 Mar;9(3):447–463. doi: 10.1016/S0006-3495(69)86396-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grissmer S., Cahalan M. D. Divalent ion trapping inside potassium channels of human T lymphocytes. J Gen Physiol. 1989 Apr;93(4):609–630. doi: 10.1085/jgp.93.4.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jan L. Y., Jan Y. N. Voltage-sensitive ion channels. Cell. 1989 Jan 13;56(1):13–25. doi: 10.1016/0092-8674(89)90979-3. [DOI] [PubMed] [Google Scholar]
  10. Kamb A., Tseng-Crank J., Tanouye M. A. Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron. 1988 Jul;1(5):421–430. doi: 10.1016/0896-6273(88)90192-4. [DOI] [PubMed] [Google Scholar]
  11. Lubin M. K+ efflux in NIH mouse 3T3 cells and transformed derivatives: dependence on extracellular Ca2+ and phorbol esters. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5097–5101. doi: 10.1073/pnas.85.14.5097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Matteson D. R., Armstrong C. M. Na and Ca channels in a transformed line of anterior pituitary cells. J Gen Physiol. 1984 Mar;83(3):371–394. doi: 10.1085/jgp.83.3.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mitani M., Harushima Y., Kuwajima K., Ikeguchi M., Sugai S. Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin. J Biol Chem. 1986 Jul 5;261(19):8824–8829. [PubMed] [Google Scholar]
  14. Neyton J., Miller C. Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+ -activated K+ channel. J Gen Physiol. 1988 Nov;92(5):569–586. doi: 10.1085/jgp.92.5.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pantoliano M. W., Whitlow M., Wood J. F., Rollence M. L., Finzel B. C., Gilliland G. L., Poulos T. L., Bryan P. N. The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case. Biochemistry. 1988 Nov 1;27(22):8311–8317. doi: 10.1021/bi00422a004. [DOI] [PubMed] [Google Scholar]
  16. Pongs O., Kecskemethy N., Müller R., Krah-Jentgens I., Baumann A., Kiltz H. H., Canal I., Llamazares S., Ferrus A. Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 1988 Apr;7(4):1087–1096. doi: 10.1002/j.1460-2075.1988.tb02917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Salkoff L., Wyman R. Genetic modification of potassium channels in Drosophila Shaker mutants. Nature. 1981 Sep 17;293(5829):228–230. doi: 10.1038/293228a0. [DOI] [PubMed] [Google Scholar]
  18. Schwarz T. L., Tempel B. L., Papazian D. M., Jan Y. N., Jan L. Y. Multiple potassium-channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature. 1988 Jan 14;331(6152):137–142. doi: 10.1038/331137a0. [DOI] [PubMed] [Google Scholar]
  19. Stühmer W., Ruppersberg J. P., Schröter K. H., Sakmann B., Stocker M., Giese K. P., Perschke A., Baumann A., Pongs O. Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J. 1989 Nov;8(11):3235–3244. doi: 10.1002/j.1460-2075.1989.tb08483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stühmer W., Stocker M., Sakmann B., Seeburg P., Baumann A., Grupe A., Pongs O. Potassium channels expressed from rat brain cDNA have delayed rectifier properties. FEBS Lett. 1988 Dec 19;242(1):199–206. doi: 10.1016/0014-5793(88)81015-9. [DOI] [PubMed] [Google Scholar]
  21. Voordouw G., Milo C., Roche R. S. Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability. Biochemistry. 1976 Aug 24;15(17):3716–3724. doi: 10.1021/bi00662a012. [DOI] [PubMed] [Google Scholar]
  22. Yokoyama S., Imoto K., Kawamura T., Higashida H., Iwabe N., Miyata T., Numa S. Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs. FEBS Lett. 1989 Dec 18;259(1):37–42. doi: 10.1016/0014-5793(89)81488-7. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES