Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel

Abstract

Summary The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+-calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1 and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C-lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition where the local conformational change induced by CaM binding at BDC2 is at the basis of channel modulation.

Eag1 K+ Channel: Endogenous Regulation and Functions in Nervous System

“…Consistently, by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by Förster resonance energy transfer (FRET), Gonçalves et al [ 43 ] found that the high affinity BD-N binding domain and the second C-terminal binding domain BD-C2 are predominantly involved in EAG1 channel inhibition by Ca 2+ . Consistently, a recent isothermal titration calorimetry (ITC) combined with crystal structure of the CaM-BDC2 complex study revealed a high affinity between CaM and BD-C2 [ 44 ]. Mutations at these two sites completely abolished CaM binding to hEAG1, indicating that the BD-N and BD-C2 binding domains are required for CaM binding.…”

hERG Function in Light of Structure

“…It is important to note that both hERG and rEAG channel structures show the S4 voltage sensor in the up position, which agrees well with the electrical field of 0 mV in the experimental setup and the open gate of the hERG channel but not with the conformational state of the closed rEAG. Like other members of the EAG subfamily, rEAG is inhibited by calmodulin in complex with Ca 2þ (CaM/Ca 2þ ) (10)(11)(12)(13)(14), and the structure provides an extraordinary view of the two proteins together. CaM/Ca 2þ either stabilizes the closed gate, forces it closed, or somehow decouples it from the VSDs while they remain in their ''up'' or activated positions.…”

Atomistic Insights of Calmodulin Gating of Complete Ion Channels

“…In any event, the take-home message is that there is a~10 Å incertitude regarding the relative position of the CaMBD. Similar to SK channels, trafficking of KCNQ channels to the membrane requires CaM [57,96,[102][103][104][105], and current density is affected by CaM availability: increases, decreases, or no effects upon CaM overexpression have been reported [106][107][108][109][110]. Since the CaMBD has a notorious aggregation tendency when produced in bacteria in the absence of CaM, it is thought that CaM is a constitutive auxiliary subunit of KCNQ channels.…”