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Positions of ?2 and ?3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

ABSTRACT: Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of ? subunits and of one of four types of modulatory ? subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ?100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ?2 and ?3, as in ?1 and ?4, TM1 is closest to ?S1 and ?S2 and TM2 is closest to ?S0. Mouse ?3 (m?3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of ?S0-?S6. We identified by elimination m?3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ?1. These are conserved in ?2-?4, which have four additional Cys (eight in total), except that m?3 has one fewer. In ?1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In m?3, the free Cys is at position 7; position 2 lacks a Cys present in all other ?2-?4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ?2-?4. Cross-linking of m?3 Cys152 to Cys substituted in the flanks of ?S0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the ?S6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of m?3 perturbed. Although the m?3 loop can move, its position with Cys152 near ?K296, in which it blocks IbTX binding, is likely favored.

SUBMITTER: Wu RS

PROVIDER: S-EPMC3536527 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Wu Roland S RS Liu Guoxia G Zakharov Sergey I SI Chudasama Neelesh N Motoike Howard H Karlin Arthur A Marx Steven O SO

The Journal of general physiology 20130101 1

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 ...[more]

PMID: 23277477

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Project description:Large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels hyperpolarize coronary artery smooth muscle cells, causing vasorelaxation. Dopamine activates BK(Ca) channels by stimulating D(1)-like receptor-mediated increases in cAMP in porcine coronary artery myocytes. There are two D(1)-like receptors (R), D(1)R and D(5)R. We hypothesize that the specific D(1)-like receptor involved in BK(Ca) channel activation in human coronary artery smooth muscle cells (HCASMCs) is the D(5)R and that activation occurs via cAMP cross-activation of cGMP-dependent protein kinase (PKG), rather than cAMP-dependent protein kinase (PKA). The effects of D(1)-like receptor agonists and antagonists on BK(Ca) channel opening in HCASMCs were examined in the presence and absence of PKG/PKA inhibition by cell-attached patch clamp. In the absence of commercially available ligands specific for D(1)R or D(5)R, D(1)R or D(5)R protein was down-regulated by transfecting HCASMCs with human D(1)R or D(5)R antisense oligonucleotides, respectively: cells transfected with scrambled oligonucleotides and nontransfected HCASMCs served as controls. The predominant ion channel conducting outward currents in nontransfected HCASMCs was identified as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel, which was activated by D(1)-like receptor agonists despite PKA inhibition with (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid (KT 5720) (300 nM), but was abolished by inhibiting PKG with 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one (KT 5823) (300 nM). D(1)-like receptor agonists activated BK(Ca) channels in all transfected cells except those transfected with D(5)R antisense oligonucleotides. Thus, the dopamine (D(1)-like) receptor mediates activation of BK(Ca) channels in HCASMCs by D(5)R, not D(1)R, and via PKG, not PKA. This is the first report of differential D(1)-like receptor regulation of vascular smooth muscle function in human cells.

Dataset Information

Positions of ?2 and ?3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Publications

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

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