Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis

Abstract

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5' and 3' rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of -90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

Ion Channels in Pulmonary Hypertension: A Therapeutic Interest?

“…They identified the critical role of CFTR as a chaperone for the trafficking of transmembrane ion channels, a mechanism which may equally pertain to other hypoxia-related lung diseases commonly associated with PH, such as COPD or lung fibrosis. In line with the notion that CFTR interacts with other ion channels, the functional complex of TASK-1/CFTR for regulation of chloride secretion in shark rectal gland has been demonstrated [ 231 ].…”

P2Y receptor regulation of K2P channels that facilitate K+ secretion by human mammary epithelial cells

“…The results of the present study demonstrate that multiple K2P channel subtypes are expressed in human mammary epithelial cells and appear to contribute to basal K ϩ secretion. At least four of these subtypes (TREK1, TREK2, TASK1 and TASK3) have been shown to be regulated by different PKC isoforms (21,29,47,52). Additionally, experiments involving BAPTA-AM pretreatment followed by UTP stimulation suggested that Ca 2ϩ -dependent and Ca 2ϩ -independent PKCs were required to produce maximum inhibition of the apical K2P current.…”

AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland (Squalus acanthias)

“…Preparation of total RNA from shark rectal gland tissue, degenerate primer design, PCR, and cloning of PCR fragments. Since the genome of the dogfish shark (Squalus acanthias) is incompletely sequenced, we sought to clone the AMPK␣ gene (PRKAA) in the shark rectal gland by preparing total RNA from rectal gland tissue, and designing degenerate primers for use in PCR reactions, and 5=and 3=-rapid amplification of cDNA ends (RACE) (32). Total RNA was extracted with TRIzol reagents (Invitrogen) from 100 mg of shark tissue that had been excised from the animal and immediately snap frozen in liquid nitrogen.…”

“…The 3=-and 5=-products were extracted from 2% agarose gels of the RACE-PCR amplifications. These PCR products were then cloned in pCR II TOPO TA cloning vectors (Invitrogen) as previously described (32). Clones were sequenced bidirectionally at the Keck Biotechnology Resource Laboratory at Yale.…”