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Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance.

ABSTRACT: Previous studies have suggested that the aspartic acid residue (D) at the third position is critical in determining the voltage polarity of fast V(j)-gating of Cx50 channels. To test whether another negatively charged residue (a glutamic acid residue, E) could fulfill the role of the D3 residue, we generated the mutant Cx50D3E. V(j)-dependent gating properties of this mutant channel were characterized by double-patch-clamp recordings in N2A cells. Macroscopically, the D3E substitution reduced the residual conductance (G(min)) to near zero and outwardly shifted the half-inactivation voltage (V(0)), which is a result of both a reduced aggregate gating charge (z) and a reduced free-energy difference between the open and closed states. Single Cx50D3E gap junction channels showed reduced unitary conductance (?(j)) of the main open state, reduced open dwell time at ±40 mV, and absence of a long-lived substate. In contrast, a G8E substitution tested to compare the effects of the E residue at the third and eighth positions did not modify the V(j)-dependent gating profile or ?(j). In summary, this study is the first that we know of to suggest that the D3 residue plays an essential role, in addition to serving as a negative-charge provider, as a critical determinant of the V(j)-dependent gating sensitivity, open-closed stability, and unitary conductance of Cx50 gap junction channels.

SUBMITTER: Xin L

PROVIDER: S-EPMC3296025 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance.

Xin Li L Nakagawa So S Tsukihara Tomitake T Bai Donglin D

Biophysical journal 20120306 5

Previous studies have suggested that the aspartic acid residue (D) at the third position is critical in determining the voltage polarity of fast V(j)-gating of Cx50 channels. To test whether another negatively charged residue (a glutamic acid residue, E) could fulfill the role of the D3 residue, we generated the mutant Cx50D3E. V(j)-dependent gating properties of this mutant channel were characterized by double-patch-clamp recordings in N2A cells. Macroscopically, the D3E substitution reduced th ...[more]

PMID: 22404924

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Project description:Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two "fast" gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (V(j)). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance gamma(h,o) and in the residual state with conductance gamma(h,res), and that both gamma(h,o) and gamma(h,res) rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of V(j) that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by gamma(h,o) and gamma(h,res) rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and V(j)-dependent gating of GJs formed from different connexin isoforms. V(j)-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive V(j) steps rising in amplitude, 2), slowly rising V(j) ramps, and 3), series of V(j) steps of high frequency. The model was used to predict V(j)-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of V(j)-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels.

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Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance.

Publications

Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance.

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