Dataset Information

Rational design of new NO and redox sensitivity into connexin26 hemichannels.

ABSTRACT: CO2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO2 (-), hemichannels comprising Cx26(K125C) can be opened by NO2 (-) and NO donors. However, NO2 (-) was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO2 (-). In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

SUBMITTER: Meigh L

PROVIDER: S-EPMC4345282 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Rational design of new NO and redox sensitivity into connexin26 hemichannels.

Meigh Louise L Cook Daniel D Zhang Jie J Dale Nicholas N

Open biology 20150201 2

CO2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanis ...[more]

PMID: 25673329

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Project description:Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca(2+) concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca(2+) regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca(2+) concentration, indicating that Ca(2+) facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca(2+)-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca(2+) insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca(2+), and (b) facilitating closing and stabilization of the closed state in the presence of Ca(2+). To explore the role of a negatively charged residue at position 50 in regulation by Ca(2+), this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)(-). D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES(-) restores the wild-type Ca(2+) regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca(2+) regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca(2+). Mutant cycle analysis supports a Ca(2+)-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca(2+) destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca(2+).

Dataset Information

Rational design of new NO and redox sensitivity into connexin26 hemichannels.

Publications

Rational design of new NO and redox sensitivity into connexin26 hemichannels.

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