Divalent cations inhibit IsK/KvLQT1 channels in excised membrane patches of strial marginal cells.
The IsK/KvLQT1 K+ channel in the apical membrane of strial marginal cells and vestibular dark cells is an essential ion transport pathway for the secretion of K+ into the endolymph of the inner ear. Study of this control point has been impeded by rundown of channel activity upon excision into commonly used cytosolic solutions. This paper describes conditions under which patches of apical membrane of strial marginal cells and vestibular dark cells from gerbil containing this channel can be excised, retaining its characteristic voltage dependence, kinetic properties, ion permeability sequence and pharmacological sensitivity, similar to those found during on-cell and perforated-patch whole cell recordings (Shen et al., Audit. Neurosci. 3 (1997) 215-230). Those excised-patch conditions include removal of Mg2+ from the cytosolic solution and use of a K+-rich pipette electrolyte. The inhibition of channel activity by Mg2+ was found to be a general feature of divalent cations; the channel was also inhibited by Ca2+, Ba2+ and Sr2+. The concentrations causing 50% inhibition of IsK/KvLQT1 channel current were 7 x 10(-5) M, 6 x 10(-6) M, 3 x 10(-4) M and 7 x 10(-5) M, respectively. It was also found that a chemical cross-linking agent, 3,3'-dithio-bis(sulfosuccinimidyl propionate) (DTSSP), which was previously shown to persistently activate IsK/KvLQTI channels expressed in Xenopus oocytes, maintained in excised patches channel activity which retained voltage dependence and pharmacological sensitivity. These data demonstrate that (1) the channel complex is inhibited by Ca2+, Mg2+ and other divalent cations, (2) the activation by Ca2+ observed previously in whole-cell preparations was due to action via other cellular pathways. These findings must be taken into account when considering the action of receptors which alter the cytosolic Ca2+ activity.
Shen
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Biophysics Laboratory, Boys Town National Research Hospital, Omaha, NE 68131, USA.
Marcus
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D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology*; D003051:Cochlea / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005849:Gerbillinae; D008274:Magnesium / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D013316:Stria Vascularis / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology
D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D026902:Potassium Channel Blockers; D013388:Succinimides; D001464:Barium; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D008274:Magnesium; D002118:Calcium