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The unique N-terminal sequence of the BKCa channel ?-subunit determines its modulation by ?-subunits.

ABSTRACT: Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the ?1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the ?1-subunit, but not by the ?2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by ?-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two ?1-?2 chimeric constructs containing the extracellular loop of ?1 or ?2, and the transmembrane and cytoplasmic domains of ?2 or ?1, respectively. Both ? chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 ?M intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of ?1 in the chimera resembled results seen with the full ?1 subunit, suggesting that the extracellular region of ?1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BKCa channels either containing or lacking this poly-serine stretch were leftward shifted by ?1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BKCa channel by ?-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.

SUBMITTER: Lorca RA

PROVIDER: S-EPMC5531486 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The unique N-terminal sequence of the BKCa channel α-subunit determines its modulation by β-subunits.

Lorca Ramón A RA Ma Xiaofeng X England Sarah K SK

PloS one 20170727 7

Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the β1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) ...[more]

PMID: 28750098

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Project description:Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two α-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total α-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 α-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of β1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of α-BKCa subunits, whereas in the absence of α-BKCa, a significant proportion of β1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of α-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total α-BKCa expression, regardless of differences in splice variant expression.

Dataset Information

The unique N-terminal sequence of the BKCa channel ?-subunit determines its modulation by ?-subunits.

Publications

The unique N-terminal sequence of the BKCa channel α-subunit determines its modulation by β-subunits.

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