Limits...
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.

Show MeSH

Related in: MedlinePlus

Modification of A43C residues by bBBr occurs at voltages that favor channel closure. (A) Macroscopic currents attributable to A43C channels are irreversibly decreased when exposed to bBBr during a voltage paradigm, steps between 0 and −90 mV, which elicits gating transitions between the open (0 mV) and either loop- and/or Vj-gating closed states (−90 mV). (B) Application of bBBr during a voltage paradigm, steps from 0 to 30 mV, which favors population of the open-channel state, has no effect on A43C macroscopic currents. The central bar in both panels indicates the time and duration for which the bath solution containing 500 µM TCEP was exchanged with the same bath solution containing 1 mM bBBr and subsequently washed with TCEP containing bath solution.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2713147&req=5

fig6: Modification of A43C residues by bBBr occurs at voltages that favor channel closure. (A) Macroscopic currents attributable to A43C channels are irreversibly decreased when exposed to bBBr during a voltage paradigm, steps between 0 and −90 mV, which elicits gating transitions between the open (0 mV) and either loop- and/or Vj-gating closed states (−90 mV). (B) Application of bBBr during a voltage paradigm, steps from 0 to 30 mV, which favors population of the open-channel state, has no effect on A43C macroscopic currents. The central bar in both panels indicates the time and duration for which the bath solution containing 500 µM TCEP was exchanged with the same bath solution containing 1 mM bBBr and subsequently washed with TCEP containing bath solution.

Mentions: Irreversible reductions in current also result when A43C channels are exposed to the polar, thiol cross-linking reagent bBBr, with voltage paradigms that favor channel closure. This is illustrated in Fig. 6 A, where bBBr was applied during a series of hyperpolarizing steps to −90 mV from a holding potential of 0 mV. A43C currents are not decreased when this reagent is applied during depolarizing steps from 0 to 30 mV (Fig. 6 B), a voltage paradigm that maintains the channel in the open state. We did not attempt to verify that the reduction in current results from cross-linking neighboring A43C residues by examining changes in fluorescence intensity that are expected to occur if bBBr cross-linked neighboring cysteine residues (Kim and Raines, 1995). Thus, it is possible that the reduction in current was due to the modification of individual thiols rather than cross-linking of neighboring thiol groups. However, regardless of the mechanism underlying the observed current reduction, the correlation between the voltage dependence of channel closure and inhibition of A43C current by bBBr indicates that A43C residues are accessible to modification only when the channel resides in a closed conformation. bBBr has no effect on Cx32*Cx43E1 currents or those present in uninjected oocytes (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)

Modification of A43C residues by bBBr occurs at voltages that favor channel closure. (A) Macroscopic currents attributable to A43C channels are irreversibly decreased when exposed to bBBr during a voltage paradigm, steps between 0 and −90 mV, which elicits gating transitions between the open (0 mV) and either loop- and/or Vj-gating closed states (−90 mV). (B) Application of bBBr during a voltage paradigm, steps from 0 to 30 mV, which favors population of the open-channel state, has no effect on A43C macroscopic currents. The central bar in both panels indicates the time and duration for which the bath solution containing 500 µM TCEP was exchanged with the same bath solution containing 1 mM bBBr and subsequently washed with TCEP containing bath solution.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2713147&req=5

fig6: Modification of A43C residues by bBBr occurs at voltages that favor channel closure. (A) Macroscopic currents attributable to A43C channels are irreversibly decreased when exposed to bBBr during a voltage paradigm, steps between 0 and −90 mV, which elicits gating transitions between the open (0 mV) and either loop- and/or Vj-gating closed states (−90 mV). (B) Application of bBBr during a voltage paradigm, steps from 0 to 30 mV, which favors population of the open-channel state, has no effect on A43C macroscopic currents. The central bar in both panels indicates the time and duration for which the bath solution containing 500 µM TCEP was exchanged with the same bath solution containing 1 mM bBBr and subsequently washed with TCEP containing bath solution.
Mentions: Irreversible reductions in current also result when A43C channels are exposed to the polar, thiol cross-linking reagent bBBr, with voltage paradigms that favor channel closure. This is illustrated in Fig. 6 A, where bBBr was applied during a series of hyperpolarizing steps to −90 mV from a holding potential of 0 mV. A43C currents are not decreased when this reagent is applied during depolarizing steps from 0 to 30 mV (Fig. 6 B), a voltage paradigm that maintains the channel in the open state. We did not attempt to verify that the reduction in current results from cross-linking neighboring A43C residues by examining changes in fluorescence intensity that are expected to occur if bBBr cross-linked neighboring cysteine residues (Kim and Raines, 1995). Thus, it is possible that the reduction in current was due to the modification of individual thiols rather than cross-linking of neighboring thiol groups. However, regardless of the mechanism underlying the observed current reduction, the correlation between the voltage dependence of channel closure and inhibition of A43C current by bBBr indicates that A43C residues are accessible to modification only when the channel resides in a closed conformation. bBBr has no effect on Cx32*Cx43E1 currents or those present in uninjected oocytes (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