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Expression of an inwardly rectifying potassium channel in Xenopus oocytes
F Périer1, K L Coulter, C M Radeke
1Department of Biological Sciences, University of California, Santa Barbara 93106.
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 +/- 0.07 microA; mean +/- 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.
Insights
Murine macrophage mRNA successfully expressed functional inwardly rectifying potassium channels in Xenopus oocytes. This finding highlights J774.1 cell line mRNA as a valuable resource for inward rectifier potassium channel cloning.
Area of Science:
- Molecular biology
- Cell biology
- Electrophysiology
Background:
- Inwardly rectifying potassium channels (Kir) are crucial for regulating membrane potential.
- Understanding Kir channel function requires efficient expression systems.
- The J774.1 murine macrophage cell line is a potential source for Kir channel mRNA.
Purpose of the Study:
- To investigate the expression of inwardly rectifying potassium channels using mRNA from the J774.1 cell line in Xenopus oocytes.
- To characterize the properties of the expressed currents.
- To evaluate the suitability of J774.1 mRNA for expression cloning of Kir channels.
Main Methods:
- Isolation and poly(A)(+)-selection of RNA from J774.1 cells.
- Size fractionation of RNA using sucrose density gradient centrifugation.
- Injection of fractionated RNA into Xenopus oocytes and subsequent electrophysiological recordings.
Main Results:
- Xenopus oocytes injected with J774.1 RNA expressed large-amplitude, inwardly rectifying potassium currents.
- The currents exhibited properties consistent with native Kir channels, including potassium selectivity and Ba2+ block.
- Optimal expression was observed with an mRNA size fraction of 4-5 kb.
- The expressed currents showed voltage dependence and extracellular potassium concentration sensitivity.
Conclusions:
- J774.1 cell line mRNA is a rich source for functional inwardly rectifying potassium channel expression.
- The expressed currents closely mimic native Kir channel characteristics.
- J774.1 mRNA represents a promising starting material for the expression cloning of Kir channel cDNAs.