In the shark rectal gland (SRG), apical Cl[superscript]- secretion through CFTR channels is tightly coupled to a basolateral K[superscript]+ conductance. The identity of this K[superscript]+ conductive pathway is unknown. Studies were performed in the isolated perfused SRG with 16 K[superscript]+ channel inhibitors at their IC50 and with acidic perfusate. During maximal chloride secretion stimulated by forskolin and IBMX, secretion was inhibited >90% by barium chloride, a non-selective inhibitor of K[superscript]+ channels. Specific inhibitors of calcium sensitive, voltage sensitive, ATP sensitive, and inward rectifying K[superscript]+ channels had no effect on chloride secretion. The inhibitors quinidine, quinine, bupivicaine, anandamide, and low perfusate pH (6.0) abruptly and reversibly inhibited secretion by >90%, consistent with the presence of the Two-Pore-Domain (4TM 2P/KCNK/K2P) family of K+ channels. Degenerate primers were designed to regions of high amino acid homology in known mammal and teleost 4TM 2P K[superscript]+ channel subtypes: TWIK, THIK, TASK, TREK, and TRAAK. PCR with cDNA from several shark tissues identified a putative TASK-1 fragment (394 bp) in shark rectal gland, brain, gill, and kidney. 5and 3 RACE PCR was used to obtain the entire 3 sequence and partial 5 sequence of the shark gene. Genome walking was then used to obtain the remaining 5sequence, including 335 bp of untranslated region sequence upstream of the start codon. The full length clone (1282bp) had an open reading frame encoding a protein of 375 amino acids. This isoform was 80% identical at the amino acid level to the human TASK-1 protein (394 amino acids). Major structural features of the human protein were conserved in the shark ortholog, including the four transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2- and long COOH-termini, and an extended extracellular loop between M1 and P1. Shark and human TASK-1 full-length clones were expressed in Xenopus oocytes and studied with two electrode voltage clamp (TEVC) techniques. Both the shark and human TASK-1 channel showed identical current voltage relationships (outward rectifying) with a reversal potential near -90 mV compared to water injected controls. The responses to the inhibitor quinine, and the TASK-1 inhibitor bupivacaine, were identical in shark and human TASK-1. However, shark TASK-1 differed from the human ortholog in two critical responses: response to pH and the metal zinc. The pKa for shark TASK-1 was 7.75 vs. 7.37 for human TASK-1, values that are exceedingly close to the arterial pH for each species, suggesting that TASK-1 channels are regulated closely by the ambient pH. An antibody specific to shark TASK-1 was generated and expression of TASK-1 protein in the rectal gland was confirmed by confocal immuno-fluorescent microscopy which revealed localization to the basolateral membrane, with some apical staining. Shark rectal gland TASK-1 appears to be the major K[superscript]+ channel coupled to secretion in the SRG, is the oldest 4TM 2P family member identified to date, and is the first TASK-1 channel identified to play an essential role in chloride secreting epithelia.

1) Both the partially euryhaline Scyliorhinus canicula and the fully euryhaline Carcharhinus leucas significantly modify plasma concentrations of urea and chloride (Cl-) (and sodium (Na+)) in response to changes in environmental salinity, in order to maintain overall plasma osmolality slightly hyper- or isosmotic to the environment. C. leucas has a greater capacity for urea retention in dilute environments. In S. canicula all of these changes occur within 12 hours of transfer, with the notable exception of increasing plasma urea in response to acute transfer to elevated salinity. 2) A new technique, 51Cr-labelled erythrocytes, was developed to assess blood volume in elasmobranch fish. S. canicula displays significant haemodilution and concentration during chronic acclimation to decreased and increased environmental salinity respectively. Significant changes in blood volume were seen within 6 hours of acute salinity transfer. 3) In vivo secretion rates were measured in the rectal gland of S. canicula during both chronic and acute salinity transfer. Significant changes in Cl- clearance occur during acute transfer, as plasma Na+ and Cl- levels are modified, but do not persist in chronically acclimated animals. This is achieved through modifications in the volume and Cl- concentration of the secretory fluid. 4) C. leucas is able to significantly alter the abundance and/or recruitment of Na+, K+-ATPase in both the rectal gland and the kidney during chronic acclimation to salinity transfer. This is presumably in response to increased requirements for NaCl secretion in SW and osmolyte retention in FW respectively. S. canicula do not significantly alter abundance and/or recruitment of Na+, K+-ATPase in the principle osmoregulatory organs following chronic acclimation to salinity transfer. 5) Chronically SW acclimated C. leucas modify the proportion of ouabain-sensitive oxygen consumption in the tissues of the rectal gland in response to the secretory endocrine stimulus C-type natriuretic peptide (CNP). No such modification occurred in the rectal glands of FW acclimated C. leucas. This represents a change in the sensitivity and response to endocrine control factors during chronic acclimation to salinity transfer in this species. No such modification was seen the in the proportion of ouabain-sensitive oxygen consumption in the rectal glands of chronically acclimated S. canicula in response to CNP. These results were discussed in relation to the capacity for modification of osmoregulatory organs in partially and fully euryhaline elasmobranchs.