Molecular cloning and expression of an inwardly rectifying K+ channel frombovine corneal endothelial cells

PURPOSE. To determine the presence of a putative inwardly rectifying K+ cha nnel in bovine corneal endothelial (BCE) cells and to characterize its mole cular and electrophysiological properties. METHODS. An RT-PCR strategy was used to clone an IRK1 channel sequence from BCE mRNA. Northern blot analysis was used to confirm expression of this se quence in cultured BCE cells. Two-electrode voltage-clamp and whole-cell pa tch-clamp recordings were used to characterize the cloned channel expressed in Xenopus oocytes and the native channels in cultured BCE cells, respecti vely. RESULTS. A full-length (1284 bp) coding sequence that shares 99.7% nucleoti de sequence and 100% amino acid sequence identity to bovine lens IRF1 (Kir2 .1) was cloned. The authors designate this sequence BCE IRK1 or BCIRK1. Nor thern blot analysis indicated that BCIRK1 mRNA is expressed in cultured BCE cells with two major transcripts of 7.5 and 5.5 kb. BCIRK1 cDNA was subclo ned into the vector, pcDNA3.1(-), and cRNA transcribed from the BCIRK1 cDNA clone was injected into Xenopus oocytes. Two-electrode voltage-clamp recor dings from injected oocytes revealed inwardly rectifying K+ currents that w ere blocked by external Ba2+ and Cs+ in a concentration- and voltage-depend ent manner. Whole-cell patch-clamp recordings from dissociated cultured BCE cells revealed strongly inwardly rectifying K+ currents with similar prope rties. CONCLUSIONS. Corneal endothelial cells express IRK1 (Kir2.1) inwardly recti fying K+ channels. Consistent with the properties of IRK1 channels, BCIRK1 is likely involved in regulating membrane potential and possibly other cell ular functions in corneal endothelial cells.