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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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Effect of 10 µM CdCl2 on heteromeric channels formed by coexpressing 1:1 mixtures of WT and A43C RNA. (A) Heteromeric channels were treated with 20 µM DTT and washed with Cs-MES, 1.8 mM Ca2+ bath solution before the application of Cd2+ shown in the trace segment. After the reduction of macroscopic currents by treatment with 10 µM CdCl2, currents were restored to 75% of pre-cadmium levels by washing with cadmium-free bath solution. The result is interpreted to indicate that 25% of channels form a high affinity cadmium site that “locks” the channel in a closed conformation (see Results). (B) Comparison of the kinetics of channel activation upon depolarization to 50 mV before (black trace), during (green trace), and after (red trace) the application of Cd2+ in the trace shown in A. The two current traces before Cd2+ were averaged, as were the final two current traces after wash. The green trace is current trace obtained just before wash with Cd2+-free solution. The similarity among normalized traces indicates that the differences in current levels are not a consequence of differences in the kinetics of activation, but most likely reflects the proportion of channels that can be activated by the voltage step.
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fig7: Effect of 10 µM CdCl2 on heteromeric channels formed by coexpressing 1:1 mixtures of WT and A43C RNA. (A) Heteromeric channels were treated with 20 µM DTT and washed with Cs-MES, 1.8 mM Ca2+ bath solution before the application of Cd2+ shown in the trace segment. After the reduction of macroscopic currents by treatment with 10 µM CdCl2, currents were restored to 75% of pre-cadmium levels by washing with cadmium-free bath solution. The result is interpreted to indicate that 25% of channels form a high affinity cadmium site that “locks” the channel in a closed conformation (see Results). (B) Comparison of the kinetics of channel activation upon depolarization to 50 mV before (black trace), during (green trace), and after (red trace) the application of Cd2+ in the trace shown in A. The two current traces before Cd2+ were averaged, as were the final two current traces after wash. The green trace is current trace obtained just before wash with Cd2+-free solution. The similarity among normalized traces indicates that the differences in current levels are not a consequence of differences in the kinetics of activation, but most likely reflects the proportion of channels that can be activated by the voltage step.

Mentions: Currents attributable to heteromeric channels formed by coinjection of equal amounts of A43C and WT Cx32*Cx43E1 RNA are less sensitive to inhibition by 10 µM Cd2+ after initial treatment with 20 µM DTT and wash with Cs-MES bath solution than currents of homomeric A43C channels. With the 1:1 RNA ratio, currents are reduced by 58 ± 14% (n = 8), suggesting that fewer cysteine residues are available to bind cadmium and/or that the affinity of cadmium binding is reduced in heteromeric channels. The large variation in effect can be explained in part by variation in the expression among oocytes of endogenous currents that are not affected by Cd2+. Notably, however, heteromeric channel currents that have been inhibited by treatment with 10 µM Cd2+ subsequent to DTT treatment and wash can be restored on average to ∼75 ± 5% (n = 4) of their initial values by washing with Cd2+-free solution (Fig. 7 A). This suggests that ∼25% of heteromeric channels bind cadmium with high affinity. This measure of recovery is not influenced by variations in the levels of endogenous currents among oocytes, and therefore provides a reliable estimate of the proportion of channels that bind Cd2+ with high affinity. As there is little or no difference in the kinetics of activation of currents in response to polarizations to 50 mV before, during, and after the application of Cd2+ (Fig. 7 B), the differences in current levels observed with the experimental paradigm used in Fig. 7 A cannot be ascribed to difference in the rates of channel activation.


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)

Effect of 10 µM CdCl2 on heteromeric channels formed by coexpressing 1:1 mixtures of WT and A43C RNA. (A) Heteromeric channels were treated with 20 µM DTT and washed with Cs-MES, 1.8 mM Ca2+ bath solution before the application of Cd2+ shown in the trace segment. After the reduction of macroscopic currents by treatment with 10 µM CdCl2, currents were restored to 75% of pre-cadmium levels by washing with cadmium-free bath solution. The result is interpreted to indicate that 25% of channels form a high affinity cadmium site that “locks” the channel in a closed conformation (see Results). (B) Comparison of the kinetics of channel activation upon depolarization to 50 mV before (black trace), during (green trace), and after (red trace) the application of Cd2+ in the trace shown in A. The two current traces before Cd2+ were averaged, as were the final two current traces after wash. The green trace is current trace obtained just before wash with Cd2+-free solution. The similarity among normalized traces indicates that the differences in current levels are not a consequence of differences in the kinetics of activation, but most likely reflects the proportion of channels that can be activated by the voltage step.
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Related In: Results  -  Collection

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fig7: Effect of 10 µM CdCl2 on heteromeric channels formed by coexpressing 1:1 mixtures of WT and A43C RNA. (A) Heteromeric channels were treated with 20 µM DTT and washed with Cs-MES, 1.8 mM Ca2+ bath solution before the application of Cd2+ shown in the trace segment. After the reduction of macroscopic currents by treatment with 10 µM CdCl2, currents were restored to 75% of pre-cadmium levels by washing with cadmium-free bath solution. The result is interpreted to indicate that 25% of channels form a high affinity cadmium site that “locks” the channel in a closed conformation (see Results). (B) Comparison of the kinetics of channel activation upon depolarization to 50 mV before (black trace), during (green trace), and after (red trace) the application of Cd2+ in the trace shown in A. The two current traces before Cd2+ were averaged, as were the final two current traces after wash. The green trace is current trace obtained just before wash with Cd2+-free solution. The similarity among normalized traces indicates that the differences in current levels are not a consequence of differences in the kinetics of activation, but most likely reflects the proportion of channels that can be activated by the voltage step.
Mentions: Currents attributable to heteromeric channels formed by coinjection of equal amounts of A43C and WT Cx32*Cx43E1 RNA are less sensitive to inhibition by 10 µM Cd2+ after initial treatment with 20 µM DTT and wash with Cs-MES bath solution than currents of homomeric A43C channels. With the 1:1 RNA ratio, currents are reduced by 58 ± 14% (n = 8), suggesting that fewer cysteine residues are available to bind cadmium and/or that the affinity of cadmium binding is reduced in heteromeric channels. The large variation in effect can be explained in part by variation in the expression among oocytes of endogenous currents that are not affected by Cd2+. Notably, however, heteromeric channel currents that have been inhibited by treatment with 10 µM Cd2+ subsequent to DTT treatment and wash can be restored on average to ∼75 ± 5% (n = 4) of their initial values by washing with Cd2+-free solution (Fig. 7 A). This suggests that ∼25% of heteromeric channels bind cadmium with high affinity. This measure of recovery is not influenced by variations in the levels of endogenous currents among oocytes, and therefore provides a reliable estimate of the proportion of channels that bind Cd2+ with high affinity. As there is little or no difference in the kinetics of activation of currents in response to polarizations to 50 mV before, during, and after the application of Cd2+ (Fig. 7 B), the differences in current levels observed with the experimental paradigm used in Fig. 7 A cannot be ascribed to difference in the rates of channel activation.

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