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The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance.

ABSTRACT: Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V(j)-gating) and the unitary gap junction channel conductance (?(j)). To better understand the molecular structure(s) determining the V(j)-gating properties and the ?(j) of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to V(j) (G(j)/V(j) relationship), slowed V(j)-gating kinetics, and reduced ?(j) as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V(j)-gating properties and ?(j) to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The G(j)/V(j) relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (G(min)) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (?(j) = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V(j)-gating and the ?(j), and the introduction of a positively charged Arg at the ninth position reduced the G(min) with a correlated disappearance of the substate at the single channel level.

SUBMITTER: Xin L

PROVIDER: S-EPMC3042583 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance.

Xin Li L Gong Xiang-Qun XQ Bai Donglin D

Biophysical journal 20101001 7

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V(j)-gating) and the unitary gap junction channel conductance (γ(j)). To better understand the molecular structure(s) determining the V(j)-gating properties and the γ(j) of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to constr ...[more]

PMID: 20923641

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Project description:Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (Vm or Vi-o). These transjunctional voltage dependent processes have been termed Vj- or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use the resulting information to derive a consensus atomic structure that is then subjected to rigorous validation. In this paper, we summarize our efforts to obtain and validate atomic models of the open and voltage-driven closed states of undocked connexin hemichannels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

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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The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance.

Publications

The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance.

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