Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias

Abstract

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

The very small-conductance K+ channel KVLQT1 and epithelial function

“…The shark isoform of K V LQT1 (sK V LQT1) is strongly expressed in the rectal gland. In X. laevis oocytes sK V LQT1 exhibits properties similar to those of mammalian K V LQT1 channels, namely voltage dependence, slow activation kinetics and 293B sensitivity [46]. In the native tissue we observed a very small K + channel (1-3 pS) but it was neither voltage-dependent nor slowly activating [5].…”

“…Their functional role and tissue distribution, however, remain unknown [23]. Several isoforms have been successfully used for the study of K V LQT1 in expression systems (species, accession number, length, reference): rat, AJ133685, 669 amino acids (aa), [13]; mouse, P97414, 604 aa, [2], shark, AJ223714, 660 aa, [46]; human, AF000571, 676 aa, [7]; and human U89364, 581 aa [28]. The respective K + currents are slowly activating at membrane voltages >-40 mV.…”

Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics

“…Until recently, chondrichthyes, which include elasmobranchs (sharks, rays and skates) were the only major line of gnathostomes, or jawed vertebrates, for which there are no major initiatives to generate genomic sequences. Sequence data from elasmobranchs have provided unique insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, G protein coupled receptors, natriuretic peptide receptors, and other biomedically relevant genes (Valentich and Forrest 1991;Henson et al 1997;Aller et al 1999;Silva et al 1999;Greger et al 1999;Waldegger et al 1999;Ke et al 2002;Wang et al 2002;Yang et al 2002;Cai et al 2001Cai et al , 2003Mattingly et al 2004b).…”

“…The specialized rectal gland of Squalus acanthias, the spiny dogfish shark, has greatly facilitated the study of cystic fibrosis, sodium and chloride secretion (Devor et al 1995;Lehrich et al 1995;Forrest 1996;Henson et al 1997;Lehrich et al 1998;Silva et al 1999;Aller et al 1999;Greger et al 1999;Waldegger et al 1999;Ke et al 2002;Yang et al 2002). Unlike many primary cell cultures that dedifferentiate and lose transport polarity immediately after isolation, primary cultures of shark rectal gland tubular cells maintain fully differentiated function and expression of all known receptors, transporters, ion channels and signal transduction pathways in vitro (Valentich and Forrest 1991;Devor et al 1995;Lehrich et al 1995;Aller et al 1999;Greger et al 1999;Waldegger et al 1999;Ke et al 2002;Yang et al 2002;Mattingly et al 2004b). A comparison of the properties of the cloned shark and human cystic fibrosis transmembrane regulator (CFTR) has provided insights into structural domains related to functional differences in the normal and mutant proteins (Marshall et al 1991).…”

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias

“…In fact, many of the fundamental principles of electrolyte secretion were identified in studies of shark rectal glands [14,15,16]. The shark homologue of the KCNQ1 was cloned recently [35]. However, to date only ROMK type K + channels have been detected in the basolateral membrane of rectal gland tubules (RGT) [9,11].…”