Molecular cloning and expression of an inwardly rectifying K(+) channel from bovine corneal endothelial cells.
OBJECTIVE
To determine the presence of a putative inwardly rectifying K(+) channel in bovine corneal endothelial (BCE) cells and to characterize its molecular 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 sequence in cultured BCE cells. Two-electrode voltage-clamp and whole-cell patch-clamp recordings were used to characterize the cloned channel expressed in Xenopus oocytes and the native channels in cultured BCE cells, respectively.


RESULTS
A full-length (1284 bp) coding sequence that shares 99.7% nucleotide sequence and 100% amino acid sequence identity to bovine lens IRK1 (Kir2.1) was cloned. The authors designate this sequence BCE IRK1 or BCIRK1. Northern 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 subcloned into the vector, pcDNA3.1(-), and cRNA transcribed from the BCIRK1 cDNA clone was injected into Xenopus oocytes. Two-electrode voltage-clamp recordings from injected oocytes revealed inwardly rectifying K(+) currents that were blocked by external Ba(2+) and Cs(+) in a concentration- and voltage-dependent manner. Whole-cell patch-clamp recordings from dissociated cultured BCE cells revealed strongly inwardly rectifying K(+) currents with similar properties.


CONCLUSIONS
Corneal endothelial cells express IRK1 (Kir2.1) inwardly rectifying K(+) channels. Consistent with the properties of IRK1 channels, BCIRK1 is likely involved in regulating membrane potential and possibly other cellular functions in corneal endothelial cells.
Yang
D
D
Departments of Cell and Developmental Biology, University of Michigan, Ann Arbor48109-0616, USA.
Sun
F
F
Thomas
L L
LL
Offord
J
J
MacCallum
D K
DK
Dawson
D C
DC
Hughes
B A
BA
Ernst
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D002586:Cesium / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004728:Endothelium, Corneal / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics*; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D017931:DNA Primers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D002586:Cesium; D001464:Barium; D011188:Potassium