Alternatively spliced domains interact to regulate BK potassium channel gating

Abstract

Most human genes contain multiple alternative splice sites believed to extend the complexity and diversity of the proteome. However, little is known about how interactions among alternative exons regulate protein function. We used the Caenorhabditis elegans slo-1 large-conductance calcium and voltage-activated potassium (BK) channel gene, which contains three alternative splice sites (A, B, and C) and encodes at least 12 splice variants, to investigate the functional consequences of alternative splicing. These splice sites enable the insertion of exons encoding part of the regulator of K + conductance (RCK)1 Ca 2+ coordination domain (exons A1 and A2) and portions of the RCK1-RCK2 linker (exons B0, B1, B2, C0, and C1). Exons A1 and A2 are used in a mutually exclusive manner and are 67% identical. The other exons can extend the RCK1-RCK2 linker by up to 41 residues. Electrophysiological recordings of all isoforms show that the A1 and A2 exons regulate activation kinetics and Ca 2+ sensitivity, but only if alternate exons are inserted at site B or C. Thus, RCK1 interacts with the RCK1-RCK2 linker, and the effect of exon variation on gating depends on the combination of alternate exons present in each isoform.Slo1 channels | regulator of K conductance domains | C. elegans A lternative splicing is observed in all eukaryotic organisms and diversifies the proteome encoded by metazoan genomes while preserving their compact sizes. Most human genes (90%) encode multiple transcripts (1, 2), and on average, each multiexon gene encodes at least seven distinct transcripts (1). Although alternative splicing is known to change protein function by adding, removing, or altering functional domains (3, 4), the combinatorial effects of alternative splicing across multiple sites on protein function have not been examined previously. To address these questions, we examined the functional differences among isoforms of the slo1 gene that is conserved among metazoans and subject to extensive alternative splicing (5, 6). The slo1 gene in Drosophila and its ortholog in humans, KCNMA1, encode 10 and 13 alternate exons, respectively (5). If all possible exon combinations were to be expressed, then each of these genes would encode more than 1,000 transcripts. By contrast, the Caenorhabditis elegans slo-1 gene is much simpler, with three alternate splice sites (7) and 12 possible splice variants. Thus, the C. elegans slo-1 gene offers an opportunity for a comprehensive analysis of the functional diversity of splice variants generated by a single gene that is conserved from worms to humans.slo1 encodes the pore-forming subunit of large-conductance, Ca 2+ -and voltage-activated K + (BK) channels. Accordingly, functional changes caused by alternative splicing are readily measured using the patch-clamp technique. BK channels regulate cellular excitability by linking Ca 2+ signaling to membrane repolarization and are essential for vascular tone (8-10), endocrine secretion (11, 12), neurotransmitter release (13, 14), and frequency tuning in hair ce...

Cell Excitability Necessary for Male Mating Behavior inCaenorhabditis elegansIs Coordinated by Interactions Between Big Current and Ether-A-Go-Go Family K+ Channels

“…Though slo-1(A2;B0;C1;D0) significantly reduces the instance of spontaneous protraction, a large percentage of the males (38%) expressing this isoform still displayed the mutant phenotype (Table 2). slo-1(A2;B0;C1;D0) is one isoform of at least 14 that exist in C. elegans (Wang et al 2001;Johnson et al 2011). We asked whether another isoform is capable of complete rescue of the mutant phenotype.…”

The BK channel: a vital link between cellular calcium and electrical signaling

“…This diversity arises in part from mutiple alternative-splice sites that result in BK channels with different amino acid sequences, which can give rise to varied apparent sensitivities to depolarization and cytoplasmic Ca 2+ in natively-expressed channels Johnson et al, 2011). In addition, BK channels in native tissues are known to co-assemble with at least two different classes of modulatory auxiliary subunits: the BK β subunits (β1-4), and a family of leucine-rich repeat containing proteins (LRRC proteins), now referred to as BK "γ" subunits (Knaus et al, 1994;Tanaka et al, 1997;Brenner et al, 2000a;Aldrich, 2010, 2012).…”

A BK (Slo1) channel journey from molecule to physiology

“…An attempt to answer this question was made taking advantage of the worm Caenorhabditis elegans. 83,84 The C. elegans BK channel gene has only 3 sites for alternative splicing (A, B, and C), and encodes 12 splice variants. 83 The splice site enable the insertion of exons encoding part of the RCK1 (exons A1 and A2) and sections of the RCK1-RCK2 linker (exons B0, Β1, Β2, C0, and C1).…”

“…83,84 The C. elegans BK channel gene has only 3 sites for alternative splicing (A, B, and C), and encodes 12 splice variants. 83 The splice site enable the insertion of exons encoding part of the RCK1 (exons A1 and A2) and sections of the RCK1-RCK2 linker (exons B0, Β1, Β2, C0, and C1). Functional studies of all isoforms show that the A1 and A2 exons regulate channel gating kinetics and Ca 2+ sensitivity but only if alternate exons are inserted in sites B or C. For example, a shift of about 40 mV in the voltage dependence arises if the variant (A1; B0; C1) is expressed compared with (A2; Β1; C0).…”