Molecular Mechanisms of the Voltage-Dependent Potentiation of KCNH Potassium Channels

Abstract

EAG-like (ELK, Kv12) voltage-gated potassium channels in the KCNH channel family are primarily and abundantly expressed in the brain. Deletion of the ELK gene leads to hyperexcitability of hippocampal neurons, epilepsy and altered cognitive functions. One structural feature of KCNH channels important for channel function is an interaction between the N-terminal EAG domain and the C-terminal cyclic nucleotide-binding homology domain (CNBHD). Here we studied an orthologous zebrafish ELK channel (zELK) using patch-clamp recording and patch-clamp fluorometry (PCF). We noticed that a depolarizing prepulse potentiated the zELK channels in both the current amplitude and the voltage sensitivity. We named this phenomenon voltage-dependent potentiation (VDP) and found it developed and recovered within hundreds of milliseconds. Using direct application of diC8-PI(4,5)P2 and specific hydrolysis of PI(4,5)P2 by rapamycin-recruitable lipid 5-phosphatase, we demonstrated that, PI(4,5)P2 inhibits zELK channels, and its depletion suppresses the VDP. In addition, the VDP can be manipulated by multiple structural perturbations including removing, mutating or replacing the intracellular N-terminal EAG domain as well as the C-terminal CNBHD. Further, combining transition metal ion FRET and incorporation of a fluorescent noncanonical amino acid L-Anap, we measured the distances between positions in the EAG domain and CNBHD during voltage-dependent channel activation and deactivation. We found there was an apparent change in the distance between the EAG domain and CNBHD with kinetics that match the on- and off-rates of VDP. This change in the distance between the N- and C-terminal regions was abolished when PI(4,5)P2 was hydrolyzed, and the simultaneous VDP was eliminated. Collectively, we propose that the activation of zELK channels involves a rearrangement of the direct interaction between the N- and C-terminal regions, which transitions the channel to a potentiated state.