Calcium-activated potassium (BK) channels are encoded by duplicate slo1 genes in teleost fishes

Abstract

Calcium-activated, large conductance potassium (BK) channels in tetrapods are encoded by a single slo1 gene, which undergoes extensive alternative splicing. Alternative splicing generates a high level of functional diversity in BK channels that contributes to the wide range of frequencies electrically tuned by the inner ear hair cells of many tetrapods. To date, the role of BK channels in hearing among teleost fishes has not been investigated at the molecular level, although teleosts account for approximately half of all extant vertebrate species. We identified slo1 genes in teleost and nonteleost fishes using polymerase chain reaction and genetic sequence databases. In contrast to tetrapods, all teleosts examined were found to express duplicate slo1 genes in the central nervous system, whereas nonteleosts that diverged prior to the teleost whole-genome duplication event express a single slo1 gene. Phylogenetic analyses further revealed that whereas other slo1 duplicates were the result of a single duplication event, an independent duplication occurred in a basal teleost (Anguilla rostrata) following the slo1 duplication in teleosts. A third, independent slo1 duplication (autotetraploidization) occurred in salmonids. Comparison of teleost slo1 genomic sequences to their tetrapod orthologue revealed a reduced number of alternative splice sites in both slo1 co-orthologues. For the teleost Porichthys notatus, a focal study species that vocalizes with maximal spectral energy in the range electrically tuned by BK channels in the inner ear, peripheral tissues show the expression of either one (e.g., vocal muscle) or both (e.g., inner ear) slo1 paralogues with important implications for both auditory and vocal physiology. Additional loss of expression of one slo1 paralogue in nonneural tissues in P. notatus suggests that slo1 duplicates were retained via subfunctionalization. Together, the results predict that teleost fish achieve a diversity of BK channel subfunction via gene duplication, rather than increased alternative splicing as witnessed for the tetrapod and invertebrate orthologue.

FIG. 1.—

Alignment of partial slo1 protein sequences from species examined. The pore region, sixth membrane-spanning domain (S6), and inner helix of the pore are labeled. Exons beginning with exon 9 are labeled above the corresponding sequence (as numbered by Beisel et al. 2007). Note that the sequence skips exon 11 because exons 10 and 11 are mutually exclusive alternatively spliced exons. The glutamic acid residues (E) within the inner helix (*) have been shown to be necessary for the high conductance of BK channels encoded by the slo1 gene. The second of these residues is replaced by an alanine in a commonly occurring splice variant (inclusion of exon 11 in place of 10) expressed in a variety of tissues in the midshipman fish Porichthys notatus. Sequences are listed by genus name in phylogenetic order after Nelson (2006).

FIG. 2.—

Relative expression of slo1a and slo1b within several tissues from the midshipman fish Porichthys notatus. Using primers designed to regions of both P. notatus slo1 genes with 100% nucleotide identify, a 266-bp fragment of both slo1 transcripts was amplified using PCR on (A) cDNA reverse transcribed from brain (brain and anterior spinal cord), auditory (saccular) epithelium, vocal muscle, trunk muscle, gill, heart, pituitary, and intestine. Half of each purified PCR product (150–350 ng, depending on tissue type) was digested with XhoI that digests only slo1b (150 and 116 bp pieces) in P. notatus as slo1a lacks an XhoI recognition site. Undigested PCR product was run on a 3% agarose gel alongside XhoI digested samples to examine the relative expression of slo1a and slo1b within each tissue. Both vocal and trunk muscles express only slo1a (100% undigested by XhoI), whereas gill and intestine express only slo1b (100% digested by XhoI). The remaining tissues expressed a mixture of both slo1a and slo1b. The expression of slo1a and slo1b is noted below the gel for each tissue. (B) The same PCR was conducted on sequenced plasmids containing either P. notatus slo1a or slo1b to produce samples that were either pure slo1a or pure slo1b. In all, 400 ng of each purified PCR product was digested with XhoI under the same reaction conditions as used in (A). As in (A), each digest was run on an agarose gel alongside an equal amount of undigested PCR product. These digests confirm that the reaction conditions used on all samples are sufficient to digest 100% of the purified slo1b sample while leaving slo1a intact.

FIG. 3.—

(A) Bayesian estimate of phylogeny for vertebrate slo1 genes. Posterior probabilities are indicated above each branch. The Ensembl and GenBank accession numbers for each sequence can be found in the Materials and Methods and tables 1 and 2. (B) A portion of a tree modified (in bold) from the Bayesian consensus tree (A) in order to resolve the polytomy between Danio rerio slo1a and the slo1a genes from all other more derived teleosts. An SH test confirmed no significant difference (P = 0.106) between the two trees and that the tree in the lower panel (B) has a more favorable likelihood score. Slo1 genes of the genes in Anguilla rostrata have been relocated (in bold) as paralogues of slo1b of other teleosts, a move favored by likelihood over the Bayesian estimate (P = 0.482, SH test). The results indicate that a teleost whole-genome duplication event was followed by loss of slo1a in anguilliforms and that a second independent duplication event led to both slo1 paralogues present in A. rostrata.

FIG. 4.—

(A) Patterns of alternative splicing in tetrapods and teleosts as seen in the organization of exons into a slo1 transcript. Sites of alternative splicing are labeled with dotted lines showing the ways in which alternatively spliced exons are included/excluded from the final transcript. Those sites present in tetrapods but absent/truncated in teleosts are shaded in gray. Asterisk (*) marks exon with variable open reading frame. Asterisks (**) mark possible stop codons. All exons and splice site numbers are after Beisel et al. (2007). (B) A representation of the membrane topology of the protein encoded by the slo1 gene of vertebrates with sites of alternative splicing indicated. The location of the membrane spanning domains (S0–S6), pore (P), intracellular hydrophobic domains (S7–S10), two regulator of potassium conductance (RCK) domains, and calcium bowl are labeled (adapted from Beisel et al. 2007).