A residue in the intracellular vestibule of the pore is critical for gating and permeation in Ca2+-activated K+ (BKCa) channels.
1. We have used patch clamp to record large-conductance Ca2+-activated K+ (BKCa) currents from a human embryonic kidney cell line (HEK293) expressing wild-type and mutant hSlo channels. 2. When we mutated F380 in the S6 region, thought to contribute to the intracellular vestibule of the pore, to isoleucine (F380I), very little channel activity was recorded. In contrast, mutation to tyrosine (F380Y) resulted in significant voltage-dependent currents. 3. The unitary conductances of F380I, F380Y and wild-type channels were 92 +/- 6 pS (n = 3), 166 +/- 5 pS (n = 3) and 294 +/- 5 pS (n = 5), respectively. 4. Both mutant and wild-type hSlo channels were sensitive to 100 nM iberiotoxin. 5. The F380Y mutant produced channels that were active at negative membrane potentials, even in the absence of Ca2+. 6. We conclude that this conserved residue within BKCa channels may line the conduction pathway and forms a key element of the gating mechanism.
Lippiat
J D
JD
Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK.
Standen
N B
NB
Davies
N W
NW
D000465:Algorithms; D019943:Amino Acid Substitution / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007668:Kidney / Q000378:metabolism; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D012604:Scorpion Venoms / Q000494:pharmacology; D014118:Toxins, Biological / Q000633:toxicity
D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D012604:Scorpion Venoms; D014118:Toxins, Biological; C064719:iberiotoxin