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Expression of an Inwardly Rectifying Potassium Channel in Xenopus Oocytes

Abstract: The murine macrophage‐like cell line J774.1 was used as a source of mRNA for the expression of inwardly rectifying potassium channels in Xenopus oocytes. RNA was isolated, poly(A)+‐selected, and size‐fractionated by sucrose density gradient centrifugation. Oocytes injected with J774.1 RNA expressed large‐amplitude current (0.9 pmn 0.07 μA; mean pmn SEM, n = 31) at ‐100 mV in 96 mM extracellular K+ showing prominent inward rectification. The inwardly rectifying currents were most strongly expressed by an mRNA size class of 4–5 kb. The expressed current displayed selectivity, conductance, and rectification properties of inwardly rectifying potassium channels in their native membranes. The current was potassium selective and was specifically blocked by Ba2+. The conductance and the voltage dependence of the current rectification depended on the extracellular potassium concentration, with the midpoint in peak conductance following the potassium equilibrium potential. This high level of expression of inward rectifier current and the absence of other expressed currents suggest that J774.1 mRNA represents an excellent starting material for expression cloning of the inward rectifier potassium channel cDNA.