Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide.
Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1 mM NBA in the bath solution, at 2.5 microM Ca2+ reduces the open probability, Po, of the channel to less than 0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca2+-sensitive, requiring 25 mM Ca2+ to raise Po to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca2+ (1 mM) protects the channels from modification. Also presented is a second class of Ca2+-activated K+ channels which are normally present in MTAL cells which open infrequently at 10 microM Ca2+ (Po = 0.01) but have a Po of 0.08 at 1 mM Ca2+. We can conclude (i) that NBA modifies the channel by shifting Ca2+-sensitivity to very high Ca2+, (ii) that NBA acts on a site involved in Ca2+ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.
D000081:Acetamides / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D002999:Clone Cells; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D007679:Kidney Medulla / Q000502:physiology*; D007700:Kinetics; D008138:Loop of Henle / Q000502:physiology; D011188:Potassium / Q000378:metabolism*; D011817:Rabbits
D000081:Acetamides; D007473:Ion Channels; C016272:N-bromoacetamide; D011188:Potassium; D002118:Calcium