Characterization of gating and peptide block of mSlo, a cloned calcium-dependent potassium channel.
The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A "triple mutant" mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism.
Sullivan
D A
DA
Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254, USA.
Holmqvist
M H
MH
Levitan
I B
IB
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004553:Electric Conductivity; D006801:Humans; D015640:Ion Channel Gating; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000494:pharmacology*; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000302:isolation & purification*; D024681:Potassium Channels, Calcium-Activated
D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D002118:Calcium