Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons

• Published in: bioRxiv
• Main Authors: Clusin, William T, Wu, Ting-Hsuan, Ling-Fang, Shi, Kao, Peter N
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 01.03.2018
• Subjects: Amino acids; Calcium channels; Calcium channels (voltage-gated); Calcium conductance; Calcium signalling; Channel gating; Cloning; Electroreceptors; Homology; Ion channels; Isoforms; mRNA; N-Terminus; Nucleotide sequence; Polymerase chain reaction; Potassium; Potassium channels (voltage-gated); Rhincodon typus; Structure-function relationships
• DOI: 10.1101/274449

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel, whose subunit is Kcnma1, a voltage-dependent calcium channel, Cacna1d, and a relatively uncharacterized K channel which interacts with the calcium channel to produce fast (20 Hz) oscillations. Large conductance calcium-activated K channels (BK) are comprised of four subunits, encoded by Kcnma1 and modulatory subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 derived from using purified mRNA of homogenized isolated electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the the Bellono et al RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent potassium channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel, Kv1.1, which is expressed at appreciable levels and seems likely to explain the 20 Hz oscillations believed to occur in vivo. The second was more similar to Kv1.5 than to Kv1.1 but was somewhat atypical. We also found a beta subunit sequence (Kcnab2) which appears not to cause fast inactivation due to specific structural features. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of the calcium activated potassium channel, Kcnma1, and found a computer generated assembly that contained a complete sequence of its beta subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 (beta1) is absent. The evolutionary origin of the newly described channels and subunits was studied by aligning skate sequences with human sequences and those found in related fish: the whale shark (R. typus) an elasmobranch, and ghost shark (C.milii). There is also homology with the lamprey, which has electroreceptors. An evolutionary tree is presented. Further research should include focusing on the subcellular locations of these channels in the receptor cells, their gating behavior, and the effects of accessory subunits on gating.