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J Struct Funct Genomics
2012 Jun 01;132:91-100. doi: 10.1007/s10969-012-9128-4.
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The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.
Kovalevskaya NV
,
Bokhovchuk FM
,
Vuister GW
.
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The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-terminal fragment of the channels (de Groot et al. in Mol Cell Biol 31:2845-2853, 12). Here, we investigate this binding in detail and find significant differences between TRPV5 and TRPV6. We also identify and characterize in vitro four other CaM binding fragments of TRPV5/6, which likely are also involved in TRPV5/6 channel regulation. The five CaM binding sites display diversity in binding modes, binding stoichiometries and binding affinities, which may fine-tune the response of the channels to varying Ca(2+)-concentrations.
Fig. 1. Topology diagram of a typical TRP channel consisting of six transmembrane domains and intracellular N- and C-termini. Predicted CaM binding sites are numbered started from C-terminal side. V5p1 and V6p1 peptides correspond to the CaM binding site 1 of TRPV5 and TRPV6, respectively, as V5p2, V5p3/V6p3, V5p4 and V5p5 correspond to the CaM binding site 2, 3, 4 and 5 of TRPV5/6 (see also Table 1)
Fig. 2. Overlaid 15N-1H-HSQC CaM-peptide NMR spectra and data analysis. Five to ten contours were deliberately spaced closely to allow for proper overlaying of the spectra and emphasis of the changes. a CaM-V5p1s spectra at CaM:V5p1s 1:0 (black), 1:1 (red) and 1:2 (blue) molar ratio. b CaM-V6p1 spectra at CaM:V6p1 1:0 (black), 1:1 (red) and 1:2 (blue) molar ratio. c Chemical shifts induced by the peptide as a function of CaM residue number at CaM-peptide 1:1 (red) and 1:2 (blue) molar ratio. d CaM-V5p1s (dark blue) and CaM-V6p1 (teal) spectra at 1:2 CaM:peptide molar ratio
Fig. 3. ITC data for the titration of CaM by V5p1s (a) and V6p1 (b). Integrated heat pulses and curves (red) fitted using “sequential binding sites model” are shown for each peptide. The resulting thermodynamic parameters are listed in Table 2
Fig. 4. Tryptophan fluorescence spectra of the peptides V5p1s (a), V6p1 (b) and V5p2 (c) in response to titration by CaM. Peptide:CaM ratios 1:0 (black), 1:0.5 (red), 1:0.75 (orange), 1:1 (green), 1:1.5 (light-blue) and 1:2 (dark blue)
Fig. 5. 15N-1H-HSQC CaM-peptide NMR spectra and data analysis. Five to ten contours were deliberately spaced closely to allow for proper overlaying of the spectra and emphasis of the changes. a CaM-V5p2 spectra at CaM:V5p1s 1:0 (black), 1:0.75 (red) and 1:1.5 (blue) molar ratio. b CaM-V5p3 spectra at CaM:V5p3 1:0 (black) and 1:1 (red) molar ratio. c CaM-V5p4 spectra at CaM:V5p4 1:0 (black), 1:1 (red), 1:2 (blue) and 1:3 (green) molar ratio. d CaM-V5p5 spectra at CaM:V5p5 1:0 (black), 1:1 (red), 1:2 (blue) and 1:3 (green) molar ratio
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