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Modes of operation of the BKCa channel beta2 subunit.

ABSTRACT: The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.

SUBMITTER: Savalli N

PROVIDER: S-EPMC2154362 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Modes of operation of the BKCa channel beta2 subunit.

Savalli Nicoletta N Kondratiev Andrei A de Quintana Sarah Buxton SB Toro Ligia L Olcese Riccardo R

The Journal of general physiology 20070701 1

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have inves ...[more]

PMID: 17591990

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Project description:BackgroundCardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.ResultsCloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.ConclusionThese studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Project description: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) are differentially modulated by the β1-subunit, but not by the β2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by β-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two β1-β2 chimeric constructs containing the extracellular loop of β1 or β2, and the transmembrane and cytoplasmic domains of β2 or β1, respectively. Both β chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 μM intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of β1 in the chimera resembled results seen with the full β1 subunit, suggesting that the extracellular region of β1 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 β1-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 β-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.

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Modes of operation of the BKCa channel beta2 subunit.

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Modes of operation of the BKCa channel beta2 subunit.

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