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Dongli Yang, Fang Sun, Lorie L. Thomas, James Offord, Donald K. MacCallum, David C. Dawson, Bret A. Hughes, Stephen A. Ernst; Molecular Cloning and Expression of an Inwardly Rectifying K+ Channel from Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(10):2936-2944.
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purpose. 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 Ba2+ 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
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
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