Abstract 416: Identification and Characterization of a Structurally Unique, Human Oxygen-Sensitive, Voltage-Gated Potassium Channel
Background: At birth, the ductus arteriosus (DA) closes in response to increased PO2 by inhibition of voltage-gated potassium channels (Kv), including Kv1.5, in the DA smooth muscle cell (DASMC). In search of DA-specific Kv1.5 splice variants, we probed human DA cDNA with validated Kv1.5 primers. This led to the serendipitous isolation of a 1.4 kb product, which we hypothesized, encoded a new human O2-sensitive K+ channel (HOSK).
Method: Sequencing revealed homology to an expressed sequence tag cloned from human dorsal root ganglia. Expression of this putative channel was assessed in a panel of human tissues by northern analysis and immunoblotting. Heterologous expression of HOSK was achieved in hypoxic Chinese Hamster Ovary (CHO) cells by transfection. Whole-cell and single-channel patch-clamp techniques were performed to assess sensitivity to O2 and H2O2. The effect of anti-HOSK antibodies or siRNA on the O2-sensitivity of human DASMC’s was measured.
Result: HOSK is encoded within the collagen1α2 gene and is structurally unique (having 4 transmembrane domains, a unique pore sequence (GVL) and a variant voltage sensor). Purification of protein and on-chip trypsin digestion using SELDI-TOF confirms the predicted amino acid sequence. HOSK transfection created a Kv current with activation/inactivation kinetics typical of Kv channels. HOSK current was inhibited (~50%) by the Kv inhibitor, 4-aminopyridine and correolide (an Kv1x inhibitor). Site-directed mutagenesis of GVL to AAA abolished HOSK-mediated K+ current. Like endogenous DASMC K+ current, HOSK current was increased by hypoxia and inhibited by O2 or H2O2. HOSK has a single channel conductance of 19 pS and isinhibited by anti-HOSK antibody. In human DASMC whole-cell current is inhibited by anti-HOSK siRNA.
Conclusion: We have identified a novel K+ channel, HOSK, from human DA. Since, HOSK is active at negative membrane potential, inhibition of HOSK may contribute to O2-induced membrane depolarization and initiation of DA closure. HOSK is a newly recognized oxygen-sensitive K+ channel with an unusual “gene within a gene” coding mechanism and a unique structure. HOSK might be crucial in tissues of the specialized oxygen-homeostatic system, especially the DA.