KCNE4 Juxtamembrane Region Interacts with Calmodulin and is Necessary for KCNQ1 Modulation

Voltage-gated potassium (KV) channels are modulated by the KCNE family of single transmembrane proteins. A membrane-based yeast two-hybrid screen to discover KCNE4 interacting proteins identified calmodulin (CaM) as a candidate. Previous studies demonstrated that CaM binding to KCNQ1 is required for functional expression of KCNQ1-KCNE1 channels in vitro, and increasing concentrations of intracellular calcium stimulate KCNQ1-KCNE1 channels in Xenopus oocytes in the presence of wild-type CaM but not mutant CaM that cannot bind calcium. We have tested the functional consequences of the interaction between KCNE4 and CaM with the hypothesis that KCNE4 modulation of KV currents may depend on its interaction with CaM. We validated the biochemical interaction between KCNE4 and CaM using CaM-agarose pull-down, and tested KCNE4 mutants that targeted putative CaM binding sites. Mutation of a juxtamembrane site (L[69-72]A) exhibited near complete disruption of CaM binding, whereas biotinylation studies performed in CHO cells confirmed expression of the mutant protein at the cell surface. The ability of L[69-72]A to modulate KCNQ1 was then studied using whole-cell patch clamp recording to determine if functional consequences accompany the loss of CaM binding. Wild-type KCNE4 completely inhibits potassium current in CHO cells transiently co-transfected with KCNQ1, but cells co-expressing KCNQ1 with L[69-72]A exhibited KCNQ1-like currents. Mean (± SEM) current density (measured during step to +60 mV from holding potential of −80 mV) in cells expressing KCNQ1 alone was 37.0 ± 4.25, not significantly different from cells co-expressing KCNQ1 with L[69-72]A (32.1 ± 3.3), but significantly different from cells co-expressing KCNQ1 with wild-type KCNE4 (3.3 ± 0.35). These studies suggest that a juxtamembrane region in KCNE4 is critical for its interaction with CaM and is necessary for modulation of KCNQ1.