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Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels.

Dodd R, Peracchia C, Stolady D, Török K - J. Biol. Chem. (2008)

Bottom Line: The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains.The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively.Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, St George's, University of London, London, SW17 0RE United Kingdom.

ABSTRACT
Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

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Ca2+ dissociation kinetics of wild type and Ca2+ binding-deficient mutant CaMs. 3 μm CaM or mutant CaM in the presence of 50 μm CaCl2 was rapidly mixed with 90 μm quin-2 in the same solution (see “Materials and Methods”) without added Ca2+ (concentrations in mixing chamber are given) at 21 °C. A, record 1, CaM, koff 10.14 ± 0.09 (S.D. of fit) s–1, ΔRF 0.076; record 2, CaM12, 10.74 ± 0.13 s–1, ΔRF 0.076. B, record 1, CaM, 10.14 ± 0.09 s–1, ΔRF 0.076; record 2, CaM34, koff 195.54 ± 7.10 s–1, ΔRF 0.045.
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fig2: Ca2+ dissociation kinetics of wild type and Ca2+ binding-deficient mutant CaMs. 3 μm CaM or mutant CaM in the presence of 50 μm CaCl2 was rapidly mixed with 90 μm quin-2 in the same solution (see “Materials and Methods”) without added Ca2+ (concentrations in mixing chamber are given) at 21 °C. A, record 1, CaM, koff 10.14 ± 0.09 (S.D. of fit) s–1, ΔRF 0.076; record 2, CaM12, 10.74 ± 0.13 s–1, ΔRF 0.076. B, record 1, CaM, 10.14 ± 0.09 s–1, ΔRF 0.076; record 2, CaM34, koff 195.54 ± 7.10 s–1, ΔRF 0.045.

Mentions: Statistical Analysis—For each data set the stopped flow kinetic experiments produced five to nine records; these records were averaged and can be seen in Figs. 2, 3, 4; for the averaged records an “S.D. fit” was determined that indicates the standard deviation of the data from the fit. From independent averages a mean was produced for each type of experiment; the number of independent averages included in the mean is displayed in the format n = number of experiments and can be found in Tables 1, 2, 3. The S.D. associated with all data under “Results” and in the tables is a measurement of the standard deviation of the mean from all the averages and is denoted by either S.D. or the ± symbol. Typically, data are presented in the format: koff value ± S.D. of mean (n = the number of independent experiments).


Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels.

Dodd R, Peracchia C, Stolady D, Török K - J. Biol. Chem. (2008)

Ca2+ dissociation kinetics of wild type and Ca2+ binding-deficient mutant CaMs. 3 μm CaM or mutant CaM in the presence of 50 μm CaCl2 was rapidly mixed with 90 μm quin-2 in the same solution (see “Materials and Methods”) without added Ca2+ (concentrations in mixing chamber are given) at 21 °C. A, record 1, CaM, koff 10.14 ± 0.09 (S.D. of fit) s–1, ΔRF 0.076; record 2, CaM12, 10.74 ± 0.13 s–1, ΔRF 0.076. B, record 1, CaM, 10.14 ± 0.09 s–1, ΔRF 0.076; record 2, CaM34, koff 195.54 ± 7.10 s–1, ΔRF 0.045.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2555998&req=5

fig2: Ca2+ dissociation kinetics of wild type and Ca2+ binding-deficient mutant CaMs. 3 μm CaM or mutant CaM in the presence of 50 μm CaCl2 was rapidly mixed with 90 μm quin-2 in the same solution (see “Materials and Methods”) without added Ca2+ (concentrations in mixing chamber are given) at 21 °C. A, record 1, CaM, koff 10.14 ± 0.09 (S.D. of fit) s–1, ΔRF 0.076; record 2, CaM12, 10.74 ± 0.13 s–1, ΔRF 0.076. B, record 1, CaM, 10.14 ± 0.09 s–1, ΔRF 0.076; record 2, CaM34, koff 195.54 ± 7.10 s–1, ΔRF 0.045.
Mentions: Statistical Analysis—For each data set the stopped flow kinetic experiments produced five to nine records; these records were averaged and can be seen in Figs. 2, 3, 4; for the averaged records an “S.D. fit” was determined that indicates the standard deviation of the data from the fit. From independent averages a mean was produced for each type of experiment; the number of independent averages included in the mean is displayed in the format n = number of experiments and can be found in Tables 1, 2, 3. The S.D. associated with all data under “Results” and in the tables is a measurement of the standard deviation of the mean from all the averages and is denoted by either S.D. or the ± symbol. Typically, data are presented in the format: koff value ± S.D. of mean (n = the number of independent experiments).

Bottom Line: The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains.The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively.Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, St George's, University of London, London, SW17 0RE United Kingdom.

ABSTRACT
Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

Show MeSH
Related in: MedlinePlus