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 c arcinoma of the cervix, is controlled by various K+- selective pores includ ing an IRK1 type inwardly rectifying K+ channel. We used the sequence previ ously reported for the human heart Kir2.1 channel to design a RT-PCR strate gy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 k b was obtained that was identical to the human cardiac Kir2.1 inward rectif ier. 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 ex pected for a Kir2.1 channel transcript was identified not only in HeLa cell s, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injecte d into Xenopus oocytes. Cell-attached and inside-out single channel recordi ngs obtained from injected oocytes provided evidence for a voltage-independ ent 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 un itary conductance for the IRK1 channel identified in HeLa cells. In additio n, the single channel conductance for inward currents, Gamma, was found to vary as a function of alpha(K), the external K+ ion activity, according to Gamma = Gamma(o) [alpha(K)](delta) with Gamma(o) = 3.3 pS and delta = 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 mu M Cs+ caused a voltage-dependent flickering, typical of a fast channel block ing process which resulted in a reduction of the channel open probability a t increasingly negative membrane potential values. The fractional electrica l distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentra tions ranging from 25 to 100 mu M caused a slow channel block, consistent w ith 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 e xpressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential.