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Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

Bily J, Grycova L, Holendova B, Jirku M, Janouskova H, Bousova K, Teisinger J - PLoS ONE (2013)

Bottom Line: Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6.The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6.This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Structures, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

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Steady-state fluorescence anisotropy measurement of interaction between TRPC6(801–878) WT and DNS- S100A1 protein in presence and absence of calcium ions.Titration of DNS-S100A1 protein (232 µM) with TRPC6 fusion protein in presence of calcium ions resulted in an increase of bound fraction (Fb) (white dots) compared to when the DNS-S100A1 protein was titrated with TRPC6 fusion protein in absence of of calcium ions (black dots). Values are expressed as the mean ± standard deviation (SD) measured from at least three independent experiments.
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pone-0062677-g004: Steady-state fluorescence anisotropy measurement of interaction between TRPC6(801–878) WT and DNS- S100A1 protein in presence and absence of calcium ions.Titration of DNS-S100A1 protein (232 µM) with TRPC6 fusion protein in presence of calcium ions resulted in an increase of bound fraction (Fb) (white dots) compared to when the DNS-S100A1 protein was titrated with TRPC6 fusion protein in absence of of calcium ions (black dots). Values are expressed as the mean ± standard deviation (SD) measured from at least three independent experiments.

Mentions: Since S100A1 is a Ca2+-binding protein and Ca2+ ions play a crucial role in TRPC6 activity regulation, the role of calcium ions in S100A1 binding to TRPC6 (801–878) was assessed. The experiment was performed in a solution without calcium. There was no increase in fluorescence anisotropy when calcium was absent (Fig. 4), suggesting that the binding is calcium-dependent. The same behavior has been detected for the CaM/TRPC6– CT (801–878) interaction [8].


Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

Bily J, Grycova L, Holendova B, Jirku M, Janouskova H, Bousova K, Teisinger J - PLoS ONE (2013)

Steady-state fluorescence anisotropy measurement of interaction between TRPC6(801–878) WT and DNS- S100A1 protein in presence and absence of calcium ions.Titration of DNS-S100A1 protein (232 µM) with TRPC6 fusion protein in presence of calcium ions resulted in an increase of bound fraction (Fb) (white dots) compared to when the DNS-S100A1 protein was titrated with TRPC6 fusion protein in absence of of calcium ions (black dots). Values are expressed as the mean ± standard deviation (SD) measured from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643951&req=5

pone-0062677-g004: Steady-state fluorescence anisotropy measurement of interaction between TRPC6(801–878) WT and DNS- S100A1 protein in presence and absence of calcium ions.Titration of DNS-S100A1 protein (232 µM) with TRPC6 fusion protein in presence of calcium ions resulted in an increase of bound fraction (Fb) (white dots) compared to when the DNS-S100A1 protein was titrated with TRPC6 fusion protein in absence of of calcium ions (black dots). Values are expressed as the mean ± standard deviation (SD) measured from at least three independent experiments.
Mentions: Since S100A1 is a Ca2+-binding protein and Ca2+ ions play a crucial role in TRPC6 activity regulation, the role of calcium ions in S100A1 binding to TRPC6 (801–878) was assessed. The experiment was performed in a solution without calcium. There was no increase in fluorescence anisotropy when calcium was absent (Fig. 4), suggesting that the binding is calcium-dependent. The same behavior has been detected for the CaM/TRPC6– CT (801–878) interaction [8].

Bottom Line: Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6.The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6.This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Structures, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

Show MeSH
Related in: MedlinePlus