Modulatory Beta Subunits Research Articles

Expression and Purification of the BK Channel Alpha-Beta1 Complex

Large conductance Ca2+ activated potassium channels (BK) are composed of pore-forming alpha-subunits and can be associated with modulatory beta-subunits. The beta1 subunit is mainly expressed in smooth muscle, and is found to increase the Ca2+ sensitivity of BK channels and slow the deactivation process. We have generated HEK293 cell lines stably expressing the hSlo alpha subunit and beta1 subunit genes. The hSlo construct carries N-terminal Flag and His tags for purification, while the mouse beta1 is fused with GFP at the C-terminus. Current recordings from inside-out patches showed a shift in the voltage sensitivity of activation consistent with co-assembly of the subunits. The alpha-beta1 complex was bound in the presence of decylmaltoside to Flag antibody affinity beads, and eluted with Flag peptide. Co-expression was verified by protein gels and western blots, and quantitative measurement of GFP fluorescence allowed the stoichiometry of the complex to be estimated.

Beta Subunits Bridge Two Alpha Subunits Within the BK Channel Tetramer

BK potassium channels contain four pore-forming alpha subunits and four modulatory beta subunits. Transmembrane (TM) helices S1-S6 of BK alpha are homologous to S1-S6 of other V-gated potassium channels; however, BK alpha contains a unique seventh TM helix, S0, N terminal to S1. The beta subunits contain two TM helices, TM1 and TM2. From the extent of endogenous disulfide crosslinking between Cys substituted for the four residues just flanking the extracellular ends of alpha S0-S6 and of beta1 TM1 and TM2, we previously inferred that the flank of S0 was closest to the four-residue loop between S3 and S4 and also contacted the flanks of S1 and S2. Furthermore, the flank of beta1 TM1 was closest to the flanks of S1 and S2, and the flank of TM2 was closest to the flank of S0. We have now extended this analysis to the membrane doman. We find that Cys in the first helical turn of S0 within the membrane forms disulfides with Cys subsituted in the first helical turns of S3 and S4 but not with similarly located Cys in S1 or S2. Thus, in the membrane, S0 is next to S3 and S4 but not to S1 and S2, although the flank of S0 reaches the flanks of S1 and S2. Furthermore, co-expression of the double-Cys mutant of alpha, W23C in the first helical turn of S0 and F144C in the S2 flank, and the double-Cys mutant of beta1, Y42C in the TM1 flank and L157C in the first helical turn of TM2, resulted in the crosslinking of two alphas through one beta1, S0 to TM2 and TM1 to S2. Thus, TM1 and TM2 of each beta subunit lie between the voltage-sensing domains (S0-S4) of adjacent alpha subunits.

Molecular investigations of BKCa channels and the modulatory β-subunits in porcine basilar and middle cerebral arteries

Large conductance calcium-activated potassium (BK(Ca)) channels are fundamental in the regulation of cerebral vascular basal tone. We investigated the expression of the mRNA transcripts for the BK(Ca) channel and its modulatory beta-subunits (beta1-beta4) in porcine basilar and middle cerebral arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BK(Ca) channel alpha-subunit and beta-subunit proteins. The BK(Ca) channel alpha-subunit RNA and protein distribution patterns were visualized using in situ hybridization and immunofluorescence studies, respectively. The study verified that the BK(Ca) channel alpha-subunit is located to smooth muscle cells of porcine basilar and middle cerebral arteries. The mRNA transcript for beta1-, beta2- and beta4-subunit were shown by RT-PCR in porcine basilar and middle cerebral arteries. However, at the protein level, only, the beta1-subunit protein was found by western blotting.

Effects of MiRP1 and DPP6 β‐subunits on the blockade induced by flecainide of KV4.3/KChIP2 channels

The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming Kv4.3 alpha-subunit, coassembled with modulatory beta-subunits as KChIP2, MiRP1 and DPP6 proteins. beta-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on Kv4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 beta-subunits. Currents were recorded in Chinese hamster ovary cells stably expressing K(V)4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. In control conditions, Kv4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an 'overshoot' in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for Kv4.3/KChIP2/DPP6 and Kv4.3/KChIP2/MiRP1/DPP6 channels were approximately 10 mV more negative than Vh values for Kv4.3/KChIP2 and Kv4.3/KChIP2/MiRP1 channels. Flecainide (0.1-100 microM) produced a similar concentration-dependent blockade of total integrated current flow (IC50 approximately 10 microM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the 'overshoot' in Kv4.3/KChIP2/MiRP1 channels. Flecainide directly binds to the Kv4.3 alpha-subunit when the channels are in the open and inactivated state and the presence of the beta-subunits modulates the blockade by altering the gating function.

Molecular cloning, tissue distribution and bioinformatics analyses of the rabbit BK channel β1 subunit gene

Large-conductance, voltage-dependent and Ca(2+)-sensitive K(+) (BK) channels are composed of pore-forming alpha subunits and the modulatory beta subunits. In smooth muscle, the modulatory beta1 subunits are vital in rendering BK channels function as an important regulator of smooth muscle tone and excitability. In this study, we cloned and characterized the BK beta1 subunit gene from rabbits (New Zealand white) and observed its tissue distribution pattern. The full-length cDNA of the BK beta1 subunit, amplified by 5'-RACE and 3'-RACE, is 1,437 bp in nucleotide containing a 447 bp 5'-UTR, a 385 bp 3'-UTR and a 576 bp open reading frame (ORF) which encodes a peptide of 191 amino acids. Sequence analyses showed that the rabbit BK beta1 subunit cDNA is 90, 84 and 82% homologous with that of human, mouse and rat respectively. The similarity is 86, 83, and 83% at the deduced amino acids level with human, mouse and rat beta1 subunit gene, respectively. Northern blots indicated that the rabbit BK beta1 subunit gene is highly expressed in sphincter of Oddi (SO) and aortal smooth muscle tissues, whereas with relatively lower level of expression in heart and skeletal muscle tissues and with no expression found in tissues of liver, lung, kidney and brain. Bioinformatics analyses indicated that the encoded protein is a membrane protein with two transmembrane helical regions containing four functional domains, one possible PKA phosphorylation site (T14) at the N-terminal and two N-glycosylation sites (N80 and N142) at the extracellular loop. For the first time, we identified and characterized the full-length cDNA sequence of the rabbit BK channel beta1 subunit gene, which will set the basis for further investigation in the transcriptional regulation of this gene.

Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

BackgroundAction potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish).ResultsWe identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3) and duplicate genes for beta4 (zbeta4.1, zbeta4.2). Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila.ConclusionThe identification of conserved orthologs to all 4 voltage-gated sodium channel beta subunit genes in zebrafish and the lack of evidence for beta subunit genes in invertebrate chordates together indicate that this gene family emerged early in vertebrate evolution, prior to the divergence of teleosts and tetrapods. The evolutionary history of sodium channel beta subunits suggests that these genes may have played a key role in the diversification and specialization of electrical signaling in early vertebrates.

MaxiK channel partners: physiological impact

The basic functional unit of the large-conductance, voltage- and Ca2+-activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore-forming alpha-subunit (MaxiKalpha) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKalpha can associate with modulatory beta-subunits (beta1-beta4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKalpha-related disorder caused by a gain-of-function C-terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKalpha interaction with its surrounding signalling partners, and its targeting to cell-specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C-terminus of MaxiKalpha are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C-terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue-specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Endothelial K+channel lacks the Ca2+sensitivity‐regulating β subunit

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.

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