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 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.
Gordon
Earl
E
Metabolic Pathways Center of Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406, USA.
Semus
Simon F
SF
Lozinskaya
Irina M
IM
Lin
Zuojun
Z
Xu
Xiaoping
X
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004553:Electric Conductivity; D007191:Indazoles / Q000494:pharmacology*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000502:physiology*
C532126:1-(1-hexyl-6-(methyloxy)-1H-indazol-3-yl)-2-methyl-1-propanone; D007191:Indazoles; D051036:Large-Conductance Calcium-Activated Potassium Channels; D013912:Threonine; D002118:Calcium