Pore Determinants of KCNQ3 K + Current Expression

Abstract

Our laboratory has suggested the pore region of KCNQ channels to be implicated in governing current amplitudes, in which large currents are possible by an interaction between a threonine and an isoleucine at the 315 and 312 positions, respectively. Consistent with this, replacement of A315 in KCNQ3 with a threonine or serine increased current amplitudes (Zaika et al., 2008. Biophys J, 95). Here, we find several mutations in KCNQ3 at position 312 (I312V, I312E and I312R) abolished the homomeric current. Co-expression of KCNQ3 I312V and I312E with wild-type (WT) KCNQ2 resulted in smaller currents vs. WT KCNQ2+3 channels, but did not modify channel voltage dependence. Evidence that the I312V and I312E mutants were expressed in the heteromers includes a shifted TEA sensitivity, compared to KCNQ2 homomers. Molecular modeling suggests the lack of current in I312V and I312E KCNQ3 channels to be due to a destabilization of the pore structure. Another lab has suggested the C-terminus KCNQ1-3 channels is critical in determining KCNQ current amplitudes (Schwake et al., 2006. J. Neurosci., 26). Moreover, the crystal structure of the KCNQ4 coiled-coil “D-helix” highlighted three positions (V619, M629 and C643) critical for KCNQ4 channels oligomerization, which are divergent in KCNQ3 (Howard et al., 2007. Neuron, 53). We find H2O2-induced oligomerization of KCNQ4 subunits, reported by native PAGE, to localize to a cysteine located at the end of the D-helix at position 643, at which only KCNQ3 possesses a histidine at the analogous position. As a probe for the role of H646 in KCNQ3 expression, we tested the H646C mutant. However, homomeric or heteromeric channels containing this mutation produced smaller currents, ruling out this divergent residue as underlying low amplitude of KCNQ3 currents. Our results confirm that the pore region is predominant in governing KCNQ channel expression.