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Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

Tang Q, Dowd TL, Verselis VK, Bargiello TA - J. Gen. Physiol. (2009)

Bottom Line: Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism.Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state.We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

View Article: PubMed Central - PubMed

Affiliation: Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

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Cadmium locks A43C unapposed hemichannels in a closed state. (A) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps from −90 to 0 mV, shown at the top of the panel. The central bar indicates the time and duration for which the bath solution containing 100 mM Cs-MES, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.6, was exchanged with the same bath solution except containing either 20 µM DTT or 10 µM CdCl2. Currents were decreased to ∼85% of maximum levels after treatment with 10 µM CdCl2. The reduction in current could only be reversed after a second exposure to 20 µM DTT. (B) 10 µM CdCl2 was applied to the channel after a long-duration hyperpolarizing step that would favor closure of both loop and Vj gates. After wash with Cs-MES bath solution, the extent of current reduction was assessed by a series of polarizing steps between −90 and 50 mV. Currents were reduced by ∼80% by Cd2+ treatment when the channels resided in a closed state. Currents could only be recovered fully after exposure to 20 µM DTT. (C) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps between 0 and 30 mV, which strongly favors population of the open-channel state. Application of 10 µM CdCl2 had no effect on the level of A43C current, indicating that A43C residues do not coordinate Cd2+ when the channel resides in the open state.
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fig5: Cadmium locks A43C unapposed hemichannels in a closed state. (A) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps from −90 to 0 mV, shown at the top of the panel. The central bar indicates the time and duration for which the bath solution containing 100 mM Cs-MES, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.6, was exchanged with the same bath solution except containing either 20 µM DTT or 10 µM CdCl2. Currents were decreased to ∼85% of maximum levels after treatment with 10 µM CdCl2. The reduction in current could only be reversed after a second exposure to 20 µM DTT. (B) 10 µM CdCl2 was applied to the channel after a long-duration hyperpolarizing step that would favor closure of both loop and Vj gates. After wash with Cs-MES bath solution, the extent of current reduction was assessed by a series of polarizing steps between −90 and 50 mV. Currents were reduced by ∼80% by Cd2+ treatment when the channels resided in a closed state. Currents could only be recovered fully after exposure to 20 µM DTT. (C) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps between 0 and 30 mV, which strongly favors population of the open-channel state. Application of 10 µM CdCl2 had no effect on the level of A43C current, indicating that A43C residues do not coordinate Cd2+ when the channel resides in the open state.

Mentions: Fig. 5 A illustrates that the inhibition of A43C currents by micromolar concentrations of Cd2+ occurs at hyperpolarizing potentials that correlate with the voltage dependence of channel closure. Membrane currents are reduced to a steady-state level, ∼85% of their DTT-treated value, after perfusion with 10 µM Cd2+ while the oocyte is repeatedly hyperpolarized by alternating steps between −90 and 0 mV. A similar reduction in current is observed when Cd2+ is applied to channels that had been closed in response to a long hyperpolarizing step to −90 mV, washed with Cd2+-free solution, and tested with polarizations alternating between −90 and +50 mV (Fig. 5 B). In contrast, the application of 10 µM Cd2+ after moderate hyperpolarization to -20 mV (not depicted), or depolarization to 30 mV (Fig. 5 C), voltages that strongly favor open-channel residency, has little if no effect on current levels. This result suggests that Cd2+ binds to A43C residues when the channel resides in a closed state. The marked reduction of A43C current by Cd2+ at large hyperpolarizing potentials can only be reversed after the application of 20 µM DTT (Fig. 5, A and B) or 10 µM TPEN (not depicted).


Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

Tang Q, Dowd TL, Verselis VK, Bargiello TA - J. Gen. Physiol. (2009)

Cadmium locks A43C unapposed hemichannels in a closed state. (A) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps from −90 to 0 mV, shown at the top of the panel. The central bar indicates the time and duration for which the bath solution containing 100 mM Cs-MES, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.6, was exchanged with the same bath solution except containing either 20 µM DTT or 10 µM CdCl2. Currents were decreased to ∼85% of maximum levels after treatment with 10 µM CdCl2. The reduction in current could only be reversed after a second exposure to 20 µM DTT. (B) 10 µM CdCl2 was applied to the channel after a long-duration hyperpolarizing step that would favor closure of both loop and Vj gates. After wash with Cs-MES bath solution, the extent of current reduction was assessed by a series of polarizing steps between −90 and 50 mV. Currents were reduced by ∼80% by Cd2+ treatment when the channels resided in a closed state. Currents could only be recovered fully after exposure to 20 µM DTT. (C) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps between 0 and 30 mV, which strongly favors population of the open-channel state. Application of 10 µM CdCl2 had no effect on the level of A43C current, indicating that A43C residues do not coordinate Cd2+ when the channel resides in the open state.
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fig5: Cadmium locks A43C unapposed hemichannels in a closed state. (A) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps from −90 to 0 mV, shown at the top of the panel. The central bar indicates the time and duration for which the bath solution containing 100 mM Cs-MES, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.6, was exchanged with the same bath solution except containing either 20 µM DTT or 10 µM CdCl2. Currents were decreased to ∼85% of maximum levels after treatment with 10 µM CdCl2. The reduction in current could only be reversed after a second exposure to 20 µM DTT. (B) 10 µM CdCl2 was applied to the channel after a long-duration hyperpolarizing step that would favor closure of both loop and Vj gates. After wash with Cs-MES bath solution, the extent of current reduction was assessed by a series of polarizing steps between −90 and 50 mV. Currents were reduced by ∼80% by Cd2+ treatment when the channels resided in a closed state. Currents could only be recovered fully after exposure to 20 µM DTT. (C) Macroscopic currents elicited from an oocyte expressing A43C unapposed hemichannels with the voltage paradigm, steps between 0 and 30 mV, which strongly favors population of the open-channel state. Application of 10 µM CdCl2 had no effect on the level of A43C current, indicating that A43C residues do not coordinate Cd2+ when the channel resides in the open state.
Mentions: Fig. 5 A illustrates that the inhibition of A43C currents by micromolar concentrations of Cd2+ occurs at hyperpolarizing potentials that correlate with the voltage dependence of channel closure. Membrane currents are reduced to a steady-state level, ∼85% of their DTT-treated value, after perfusion with 10 µM Cd2+ while the oocyte is repeatedly hyperpolarized by alternating steps between −90 and 0 mV. A similar reduction in current is observed when Cd2+ is applied to channels that had been closed in response to a long hyperpolarizing step to −90 mV, washed with Cd2+-free solution, and tested with polarizations alternating between −90 and +50 mV (Fig. 5 B). In contrast, the application of 10 µM Cd2+ after moderate hyperpolarization to -20 mV (not depicted), or depolarization to 30 mV (Fig. 5 C), voltages that strongly favor open-channel residency, has little if no effect on current levels. This result suggests that Cd2+ binds to A43C residues when the channel resides in a closed state. The marked reduction of A43C current by Cd2+ at large hyperpolarizing potentials can only be reversed after the application of 20 µM DTT (Fig. 5, A and B) or 10 µM TPEN (not depicted).

Bottom Line: Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism.Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state.We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

View Article: PubMed Central - PubMed

Affiliation: Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

Show MeSH
Related in: MedlinePlus