An Arrhythmic Mutation E7K Facilitates TRPM4 Channel Activation via Enhanced PIP(2) Interaction

A Ca(2+)-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 (Glu(5) to Lys(5); 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 PIP(2) (diC(8)PIP(2)) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (K(d)) of diC(8)PIP(2) for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 μM). FRET-based PIP(2) measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP(2) level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent K(d) values estimated from DrVSP-mediated PIP(2) depletion: 0.97 and 1.06 μM, respectively), being associated with the differential PIP(2)-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 PIP(2) 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 PIP(2) interaction may account for the arrhythmogenic potential of this mutation.