The potassium channel KCNQ4, expressed in the mammalian cochlea, has been associated tentatively with an outer hair cell (OHC) potassium current, I(K,n), a current distinguished by an activation curve shifted to exceptionally negative potentials. Using CHO cells as a mammalian expression system, we have examined the properties of KCNQ4 channels under different phosphorylation conditions. The expressed current showed the typical KCNQ4 voltage-dependence, with a voltage for half-maximal activation (V(1/2)) of -25 mV, and was blocked almost completely by 200 microM linopirdine. Application of 8-bromo-cAMP or the catalytic sub-unit of PKA shifted V(1/2) by approximately -10 and -20 mV, respectively. Co-expression of KCNQ4 and prestin, the OHC motor protein, altered the voltage activation by a further -15 mV. Currents recorded with less than 1 nM Ca(2+) in the pipette ran down slowly (12% over 5 min). Buffering the pipette Ca(2+) to 100 nM increased the run-down rate sevenfold. Exogenous PKA in the pipette prevented the effect of elevated [Ca(2+)](i) on run-down. Inhibition of the calcium binding proteins calmodulin or calcineurin by W-7 or cyclosporin A, respectively, also prevented the calcium-dependent rapid run-down. We suggest that KCNQ4 phosphorylation via PKA and coupling to a complex that may include prestin can lead to the negative activation and the negative resting potential found in adult OHCs.