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

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 Ca21 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 voltagedependent 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.needed to maintain the native structure of voltage-dependent K+ channels. According to this picture, Ca2+ removal partially denatures the channel so that its pore loses the ability to discriminate among small ions and its voltage-dependent "gate" is unable to close.One problem that makes this interpretation equivocal is that the neuronal membrane is electrically complicated, providing a home for many different types of ion channels. The experiments in neurons do not allow us to conclude with certainty that the leak induced by Ca2+ removal is in fact mediated by the specific set of voltage-dependent K+ channels under study, rather than by other membrane proteins present. The experiments here are designed to test this idea rigorously; we examine the effect of Ca2+ removal in a non-neuronal cell line in which a heterologous voltagedependent K+ channel is specifically expressed by infection with a recombinant baculovirus. The results clearly demonstrate that the nonselective leak induced by removing Ca2+ has its origin in a massive functional alteration of the expressed K+ channels.Voltage-dependent K+ channels play a central role in the generation, propagation, and integration of electrical signals in many types of cells. These channels all display high selectivity for K+ over Na+ and strong voltage dependence of opening and closing rates. The long-recognized functional similarities among voltage-dependent K+ channels are now being rationalized in terms of homologies among members of a very large molecular family (1-5). Because membrane voltage is the physiological signal used to switch these channels between open and closed conformations, relatively little attention has been given to the roles of physiological ligands in their proper functioning. In particular, Ca2+, which is crucially involved in the action of Ca2+-activated K+ channels and many membrane-transducing systems, is not generally viewed as being dir...

Effect of external cation concentration and metabolic inhibitors on membrane potential of human glial cells.

“…The close connection between decreased K+ conductance and increased leak conductance was recently demonstrated by Armstrong & Miller (1990) in an insect cell line which had been infected with virus carrying cDNA for Drosophila shaker A-type K+ channels. The effect was augmented in solutions that were both Ca2+ and K+ free.…”

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“…On present evidence, membrane strength is thus distinct from membrane ionic permeability, where some proteinaceous membrane channels require [CaW+]. to retain their normal properties (Almers, McCleskey & Palade, 1984;Armstrong & Miller, 1990). As to the small reduction in membrane…”

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