Characterizing the role of Thr352 in the inhibition of the large conductance Ca2+-activated K+ channels by 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone

Large conductance Ca(2+)-activated K(+) (BK) channels are known to be regulated by both intracellular Ca(2+) and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and...

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Bibliographic Details
Published in:The Journal of pharmacology and experimental therapeutics 2010-08, Vol.334 (2), p.402-409
Main Authors: Gordon, Earl, Semus, Simon F, Lozinskaya, Irina M, Lin, Zuojun, Xu, Xiaoping
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Calcium - physiology
CHO Cells
Cricetinae
Cricetulus
Electric Conductivity
Indazoles - pharmacology
Large-Conductance Calcium-Activated Potassium Channels - antagonists & inhibitors
Large-Conductance Calcium-Activated Potassium Channels - genetics
Large-Conductance Calcium-Activated Potassium Channels - physiology
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Patch-Clamp Techniques
Sequence Homology, Amino Acid
Threonine - physiology
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Summary:Large conductance Ca(2+)-activated K(+) (BK) channels are known to be regulated by both intracellular Ca(2+) and voltage. Although BK channel modulators have been identified, there is a paucity of information regarding the molecular entities of this channel that govern interaction with blockers and activators. Using both whole-cell and single-channel electrophysiological studies we have characterized the possible role that a threonine residue in the pore region of the channel has on function and interaction with BK channel modulators. A threonine-to-serine substitution at position 352 (T352S) resulted in a 59-mV leftward shift in the voltage-dependent activation curve. Single-channel conductance was 236 pS for the wild-type channel and 100 pS for the T352S mutant, measured over the range -80 mV to +80 mV. In addition, there was an almost 10-fold reduction in the potency of the BK channel inhibitor 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP), the IC(50) values being 4.3 +/- 0.3 and 38.2 +/- 3.3 nM for wild-type and mutant channel, respectively. There was no significant difference between wild type and the mutant channel in response to inhibition by iberiotoxin. The IC(50) was 8.1 +/- 0.3 nM for the wild type and 7.7 +/- 0.3 nM for the mutant channel. Here, we have identified a residue in the pore region of the BK channel that alters voltage sensitivity and reduces the potency of the blocker HMIMP.
ISSN:1521-0103
DOI:10.1124/jpet.110.166017