An Arrhythmic Mutation E7K Facilitates TRPM4 Channel Activation via Enhanced PIP2 Interaction

Abstract

A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5; E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 μM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 μM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the ‘WT’ or ‘E7K’ sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.

Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

“…TRP channels are a large superfamily of proteins expressed in several tissues where they are involved in diverse signaling processes and in disease [3]. Among the various TRP channels expressed in the cardiovascular system, canonical (e.g., TRPC1 and C3-C7), melastatin (e.g., TRPM4 and M7), vanilloid (e.g., TRPV1 and V2), and polycystin (e.g., TRPP1/2) channels are present in the heart and are implicated in the physiological functions and in cardiac abnormalities such as arrhythmogenesis and heart failure [4,5]. The role of cardiac TRP channels has been explored in fibroblasts and in pacemaker cells (see reference [3]) but much less in cardiomyocytes.…”

Theoretical Investigation of the Mechanism by which A Gain-of-Function Mutation of the TRPM4 Channel Causes Conduction Block

“…Furthermore, in our numerical model simulations, simple doubling of the maximal wild-type TRPM4 channel activity produced only marginal changes in the resting membrane potential and the shape of AP [13]. In addition, our recent study disclosed the functional abnormality of E7K mutation that unusually strengthens the interaction of TRPM4 channel activity with endogenous PIP 2 thereby increasing the risk of generating triggered activities [14]. These facts raise the question of whether additional functional changes may also be involved in the pathogenesis of E7K-associated conduction disorders.…”

TRPM7 N-terminal region forms complexes with calcium binding proteins CaM and S100A1

“…We identified a new TRPM7np T523-L535 binding region revealing the ability to bind CaM and S100A1. Our biophysical and computational characterization of TRPM7np complexes with modulatory ligands CaM and S100A1 may help to elucidate the mechanism of TRPM7 channel function [ 54 ]. The structural resolution from CryoEM analysis revealed an accessible TRPM7np binding region located throughout the TRPM7 channel.…”