Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4.
Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.
Lancaster
M K
MK
School of Biomedical Sciences and Biochemistry & Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. m.k.lancaster@leeds.ac.uk
Dibb
K M
KM
Quinn
C C
CC
Leach
R
R
Lee
J K
JK
Findlay
J B
JB
Boyett
M R
MR
D000818:Animals; D001464:Barium / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008845:Microinjections; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011073:Polyamines / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D014981:Xenopus
D018076:DNA, Complementary; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D011073:Polyamines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D001464:Barium; D008274:Magnesium