Molecular Characterization of an Inwardly Rectifying K+ Channel from HeLa Cells

Molecular Characterization of an Inwardly Rectifying K+ Channel from HeLa Cells Previous patch-clamp studies have shown that the potassium permeability of the plasma membrane in HeLa cells, a cell line derived from an epidermoid carcinoma of the cervix, is controlled by various K+-selective pores including an IRK1 type inwardly rectifying K+ channel. We used the sequence previously reported for the human heart Kir2.1 channel to design a RT-PCR strategy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 kb was obtained that was identical to the human cardiac Kir2.1 inward rectifier. The nature of the cloned channel was also confirmed in a Northern blot analysis where a signal of 5.3 kb corresponding to the molecular weight expected for a Kir2.1 channel transcript was identified not only in HeLa cells, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injected into Xenopus oocytes. Cell-attached and inside-out single channel recordings obtained from injected oocytes provided evidence for a voltage-independent K+-selective channel with current/voltage characteristics typical of a strong inward rectifier. The single channel conductance for inward currents measured in 200 mm K2SO4 conditions was estimated at 40 ± 1 pS (n= 3), for applied voltages ranging from −100 to −160 mV, in agreement with the unitary conductance for the IRK1 channel identified in HeLa cells. In addition, the single channel conductance for inward currents, Γ, was found to vary as a function of αK, the external K+ ion activity, according to Γ=Γ0 [αK]δ with Γ0= 3.3 pS and δ= 0.5. Single channel recordings from injected oocytes also provided evidence of a voltage-dependent block by external Cs+ and Ba2+. The presence of 500 μm Cs+ caused a voltage-dependent flickering, typical of a fast channel blocking process which resulted in a reduction of the channel open probability at increasingly negative membrane potential values. The fractional electrical distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentrations ranging from 25 to 100 μm caused a slow channel block, consistent with the binding of a single Ba2+ ion at a site located at half the membrane span. It is concluded on the basis of these observations that HeLa cells expressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Molecular Characterization of an Inwardly Rectifying K+ Channel from HeLa Cells

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1999 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900470
Publisher site
See Article on Publisher Site

Abstract

Previous patch-clamp studies have shown that the potassium permeability of the plasma membrane in HeLa cells, a cell line derived from an epidermoid carcinoma of the cervix, is controlled by various K+-selective pores including an IRK1 type inwardly rectifying K+ channel. We used the sequence previously reported for the human heart Kir2.1 channel to design a RT-PCR strategy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 kb was obtained that was identical to the human cardiac Kir2.1 inward rectifier. The nature of the cloned channel was also confirmed in a Northern blot analysis where a signal of 5.3 kb corresponding to the molecular weight expected for a Kir2.1 channel transcript was identified not only in HeLa cells, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injected into Xenopus oocytes. Cell-attached and inside-out single channel recordings obtained from injected oocytes provided evidence for a voltage-independent K+-selective channel with current/voltage characteristics typical of a strong inward rectifier. The single channel conductance for inward currents measured in 200 mm K2SO4 conditions was estimated at 40 ± 1 pS (n= 3), for applied voltages ranging from −100 to −160 mV, in agreement with the unitary conductance for the IRK1 channel identified in HeLa cells. In addition, the single channel conductance for inward currents, Γ, was found to vary as a function of αK, the external K+ ion activity, according to Γ=Γ0 [αK]δ with Γ0= 3.3 pS and δ= 0.5. Single channel recordings from injected oocytes also provided evidence of a voltage-dependent block by external Cs+ and Ba2+. The presence of 500 μm Cs+ caused a voltage-dependent flickering, typical of a fast channel blocking process which resulted in a reduction of the channel open probability at increasingly negative membrane potential values. The fractional electrical distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentrations ranging from 25 to 100 μm caused a slow channel block, consistent with the binding of a single Ba2+ ion at a site located at half the membrane span. It is concluded on the basis of these observations that HeLa cells expressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Jan 1, 1999

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