The Role of the Cyclic Nucleotide Binding Homology Domain in Voltage and Calcium Dependent Gating of EAG Potassium Channels

Abstract

Ether-a-go-go (EAG or KCNH1) potassium channels are inhibited by increases of intracellular calcium concentration [Ca2+]i through a calmodulin (CaM) mediated mechanism. Three CaM binding sites have already been identified on intracellular domains, one close to the N-terminally located eag-domain and two close to the cyclic nucleotide binding homology domain (cNBHD) on the C-terminus. Crystal structures of the cNBHD have revealed that instead of binding cyclic nucleotides the binding pocket is occupied by a short β-strand (intrinsic ligand). In this study we set out to elucidate the role of the cNBHD and intrinsic ligand in CaM regulation of these channels. hEAG1 currents were recorded in Xenopus oocytes using two electrode voltage clamp. Elevation of [Ca2+]i by application of the Ca2+-ionophore ionomycin (5μM) and SERCA inhibitor thapsigargin (5μM) profoundly inhibited wild-type (WT) hEAG1 currents in a manner that was blocked by the CaM binding peptide mastoparan. Mutation of the intrinsic ligand slowed the time to 80% activation at +40 mV (t80%,act) from 256.35 ± 31.05 ms (n=6) in WT hEAG1 to 1046 ± 15.62 ms (n=3) in Y672A:L674A hEAG1. Substitution of binding pocket residues with large residues also caused similar slowing of t80%,act (e.g. 682.6 ± 17.9 ms for A609L, n=3). Despite this, none of these mutations caused a reduction in Ca2+-sensitivity. However, deletion of the cNBHD abolished the response to elevated [Ca2+]i and also had a profound effect on channel gating (t80%,act 426.6 ± 44.67 ms, n=3). Overall, our results suggest that an intact cNBHD is required for Ca2+-dependent inhibition, but reducing the affinity of the intrinsic ligand for its binding pocket is important for gating not Ca2+-sensitivity.