Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4.

Kristyna Bousova,Jiri Vondrasek,Monika Zouharova,Veronika Vetyskova,Ivan Barvik,Petr Herman,Pavel Majer,Kateřina Hofbauerová,Klara Postulkova,Lenka Monincova

doi:10.3390/ijms21124323

Abstract

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Highlights

Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4) is a nonselective monovalent cation channel that is activated and subsequently blocked by intracellular calcium (Ca2+) [1] at negative plasma membrane potentials [2,3,4]
We have found binding epitopes at the N- and C-termini of transient receptor potential channels (TRPs) cation channel subfamily melastatin member 4 (TRPM4) shared by CaM, S100A1 and PIP2
Solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4

Summary

Introduction

Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4) is a nonselective monovalent cation channel that is activated and subsequently blocked by intracellular calcium (Ca2+) [1] at negative plasma membrane potentials [2,3,4]. Specific mutations in the TRPM4 gene lead to the inhibition of the channel function that directly causes familial cases of heart block disease [5]. Eight structures of the TRPM4 channel have been solved using single-particle cryo-Electron Microscopy (EM) at an overall resolution ranging from 2.9 to 3.8 Å [8,9,10,11]. The TRPM4 structural topology represents a crown-like tetrameric transmembrane core with a domain-swapped architecture [9]. The channel-gating can be allosterically influenced by endogenous modulators that bind to the intracellular tails of TRPM4. The ultimate Ca2+ sensitivity is strongly regulated by several intracellular factors, including calmodulin (CaM), phosphatidylinositol 4, 5-bisphosphate (PIP2), protein kinase C, ATP, etc. [1,12,13,14]

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