Cloning and functional expression of rKCNQ2 K+ channel from rat brain

By homologue cloning, we have isolated a cDNA encoding a voltage-gated K+ c hannel, rKCNQ2, from a rat brain cDNA library using RACE. The open reading frame of the translated protein comprises 852 amino acids with 6 transmembr ane segments and a pore motif between S5 and SO. rKCNQ2 shares 96% amino ac id identity with human KCNQ2 in which mutations cause a form of epilepsy kn own as benign familial neonatal convulsions (BFNC). Northern blotting with a rKCNQ2-specific probe revealed a robust single band of 8.6-kb transcript expressed in brain not in other tissues. Functional expression of rKCNQ2 in an HEK 293 cell line by whole-cell current recordings and in Xenopus oocyt es by two-electrode voltage clamp showed outward K+ selective currents that displayed delayed rectifier-type kinetics. The G-Ii curve, fitted with a B oltzmann function, showed voltage dependence of activation with a threshold of activation approximately -60 mV. The rKCNQ2 currents were sensitive to TEA block with a Ki of 0.1 mM. In addition, rKCNQ2 currents were down-regul ated upon exposure of cells to either a broad-spectrum tyrosine kinase inhi bitor genistein or a Src-like tyrosine kinase inhibitor herbimycin A. Our f indings add a rodent member to the KCNQ channel subfamily, providing new in formation of the channel modulation, and will facilitate generation of rode nt models of epilepsy. (C) 2000 Elsevier Science B.V. All rights reserved.