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

D. Yang, F. Sun, L. L. Thomas, J. Offord, D. K. Macallum, D. C. Dawson, B. A. Hughes, S. A. Ernst

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 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.

Original languageEnglish (US)
Pages (from-to)2936-2944
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume41
Issue number10
StatePublished - 2000
Externally publishedYes

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Inwardly Rectifying Potassium Channel
Molecular Cloning
Endothelial Cells
Oocytes
Xenopus
Northern Blotting
Electrodes
Complementary DNA
Clone Cells
Complementary RNA
Messenger RNA
Membrane Potentials
Lenses
Amino Acid Sequence
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Yang, D., Sun, F., Thomas, L. L., Offord, J., Macallum, D. K., Dawson, D. C., ... Ernst, S. A. (2000). Molecular cloning and expression of an inwardly rectifying K+ channel from bovine corneal endothelial cells. Investigative Ophthalmology and Visual Science, 41(10), 2936-2944.

Molecular cloning and expression of an inwardly rectifying K+ channel from bovine corneal endothelial cells. / Yang, D.; Sun, F.; Thomas, L. L.; Offord, J.; Macallum, D. K.; Dawson, D. C.; Hughes, B. A.; Ernst, S. A.

In: Investigative Ophthalmology and Visual Science, Vol. 41, No. 10, 2000, p. 2936-2944.

Research output: Contribution to journalArticle

Yang, D, Sun, F, Thomas, LL, Offord, J, Macallum, DK, Dawson, DC, Hughes, BA & Ernst, SA 2000, 'Molecular cloning and expression of an inwardly rectifying K+ channel from bovine corneal endothelial cells', Investigative Ophthalmology and Visual Science, vol. 41, no. 10, pp. 2936-2944.
Yang, D. ; Sun, F. ; Thomas, L. L. ; Offord, J. ; Macallum, D. K. ; Dawson, D. C. ; Hughes, B. A. ; Ernst, S. A. / Molecular cloning and expression of an inwardly rectifying K+ channel from bovine corneal endothelial cells. In: Investigative Ophthalmology and Visual Science. 2000 ; Vol. 41, No. 10. pp. 2936-2944.
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T1 - Molecular cloning and expression of an inwardly rectifying K+ channel from bovine corneal endothelial cells

AU - Yang, D.

AU - Sun, F.

AU - Thomas, L. L.

AU - Offord, J.

AU - Macallum, D. K.

AU - Dawson, D. C.

AU - Hughes, B. A.

AU - Ernst, S. A.

PY - 2000

Y1 - 2000

N2 - 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 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.

AB - 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 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.

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