TRPM6 N-Terminal CaM- and S100A1-Binding Domains

Abstract

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member—the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

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

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Figure 4 Multiple alignment of selected CaM binding regions in TRPM channels. CLUSTAL 1.2.4 multiple sequence alignment of CaM/S100A1 binding regions of TRPMs N-termini [ 15 , 25 , 37 , 38 , 40 , 41 ]. The sequence alignment shows a first perfect match (indicated by an asterisk) of arginine residues and second almost accurate match (indicated by dots) of basic residues across the TRPM sequences.

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“…Specifically, the negatively charged residues of CaM involved in TRPM7np interactions were identified as: E6, E11, E14, E114 and E120 which correspond to CaM negative residues involved in the interactions in TRPV6/CaM complex [ 44 ]. The S100A1 residues D46, D52, N86 involved in TRPM7np interactions correspond to the negative cluster of S100A1 involved at TRPM6np/S100A1 complex formation [ 15 ]. The ligand docking supported the proposal of high participation of TRPM7np R525, R530, K531, R532 and R534 basic residues in the complex formations with CaM and S100A1 as predicted by multiple sequence alignment of TRPM binding regions.…”

“…The multiple sequence alignment analysis of TRPM binding regions revealed consensus sequences of basic amino acid residues (RxxxxR/K, where x is any amino acid) in analysed regions (Figure 4) suggested the importance of R525, R530, K531, R532 and R534 in TRPM7np. This analysis of multiple TRPM sequences was [15,25,37,38,40,41]. The sequence alignment shows a first perfect match (indicated by an asterisk) of arginine residues and second almost accurate match (indicated by dots) of basic residues across the TRPM sequences.…”

“…Possible direct or indirect and more complex regulation of TRPM7 by CaM/Ca 2+ still needs to be explored. The closest member to TRPM7, the TRPM6 member has already revealed overlapping binding epitopes for CaM and S100A1 at the N-termini [ 15 ].…”

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“…TRPs channels can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). A CBP such as the S100 calcium-binding protein A1 (S100A1), is known for its modulatory activities toward receptors [ 47 ].…”