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.
Highlights
Despite steady advancements in prevention and treatment in the past few decades, cardiac arrhythmias remain among the leading causes of mortality in developed countries [1]
To confirm the previous finding that PI(4,5)P2 is essential for TRPM4 channel activity [12,13], and to explore whether there is any difference in PIP2 sensitivity between its wild type (WT) and E7K mutant, we performed single-channel recordings of TRPM4 channels heterologously expressed in Human embryonic kidney cells 293 (HEK293) cells
The extent of reactivation was well described by a Hill-type equation with apparent EC50 values of 2.40 ± 0.23 μM (n = 5) and 1.44 ± 0.20 μM (n = 5) for the WT- and E7K-TRPM4 channels, respectively (Figure 1C). These results indicate that the E7K mutant is more potently reactivated by diC8-PIP2 than its WT
Summary
Despite steady advancements in prevention and treatment in the past few decades, cardiac arrhythmias remain among the leading causes of mortality in developed countries [1]. Comprehensive understanding their pathogenesis continues to be an important goal with the highest priority. The N-terminal mutation c.19G → A in the TRPM4 gene (i.e., p.7E → K) was found in a few pedigrees of patients manifesting progressive conduction blocks and was associated with sudden death This mutation was shown to increase the cell-surface expression of the TRPM4 channel protein via impaired SUMOylation in vitro, and may cause a depolarizing shift of the resting membrane potential [6]. This would in turn facilitate the inactivation of the voltage-dependent Na+ channels and presumably induce cell death, which may account for the observed fibrotic degeneration of Purkinje fibers, leading to conduction failures [7]
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