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

By homologue cloning, we have isolated a cDNA encoding a voltage-gated K(+) channel, rKCNQ2, from a rat brain cDNA library using RACE. The open reading frame of the translated protein comprises 852 amino acids with 6 transmembrane segments and a pore motif between S5 and S6. rKCNQ2 shares 96% amino acid identity with human KCNQ2 in which mutations cause a form of epilepsy known 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 recording and in Xenopus oocytes by two-electrode voltage clamp showed outward K(+) selective currents that displayed delayed rectifier-type kinetics. The G-V curve, fitted with a Boltzmann 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-regulated upon exposure of cells to either a broad-spectrum tyrosine kinase inhibitor genistein or a Src-like tyrosine kinase inhibitor herbimycin A. Our findings add a rodent member to the KCNQ channel subfamily, providing new information of the channel modulation, and will facilitate generation of rodent models of epilepsy.