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Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.

Kronengold J, Trexler EB, Bukauskas FF, Bargiello TA, Verselis VK - J. Gen. Physiol. (2003)

Bottom Line: Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified.The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits.If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA.

ABSTRACT
Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

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Residues in E1 of Cx46 near the TM1 border of Cx46 are important for the effects caused by the Cx46*32E1 substitution. (A) Sequence comparison of E1 domains of Cx46, Cx32, and Cx43. The accepted topology of a connexin subunit has four transmembrane (TM) domains, extracellular E1 and E2 loops, and cytoplasmic NH2-terminal (NT), loop (CL), and carboxy terminal (CT) domains. E42 resides at the TM1/E1 border and R76 at the E1/TM2 border. (B) Representative recordings of Cx46, Cx46*32E1, and Cx46*32E1(K49Q + S51D) hemichannel currents in Xenopus oocytes obtained with 8-s voltage ramps from −70 to +70 mV applied to cell-attached patches containing single hemichannels. The solid gray lines represent exponential fits to the open-state current. Substitution of the E1 domain of Cx32 into Cx46 (Cx46*32E1) substantially reduced unitary conductance and converted open-channel current rectification from inward to outward. Restoring the residues at positions 49 and 51 in the Cx46*32E1 chimera back to Cx46 sequence largely restored wild-type Cx46 properties.
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fig1: Residues in E1 of Cx46 near the TM1 border of Cx46 are important for the effects caused by the Cx46*32E1 substitution. (A) Sequence comparison of E1 domains of Cx46, Cx32, and Cx43. The accepted topology of a connexin subunit has four transmembrane (TM) domains, extracellular E1 and E2 loops, and cytoplasmic NH2-terminal (NT), loop (CL), and carboxy terminal (CT) domains. E42 resides at the TM1/E1 border and R76 at the E1/TM2 border. (B) Representative recordings of Cx46, Cx46*32E1, and Cx46*32E1(K49Q + S51D) hemichannel currents in Xenopus oocytes obtained with 8-s voltage ramps from −70 to +70 mV applied to cell-attached patches containing single hemichannels. The solid gray lines represent exponential fits to the open-state current. Substitution of the E1 domain of Cx32 into Cx46 (Cx46*32E1) substantially reduced unitary conductance and converted open-channel current rectification from inward to outward. Restoring the residues at positions 49 and 51 in the Cx46*32E1 chimera back to Cx46 sequence largely restored wild-type Cx46 properties.

Mentions: Substitution of the E1 domain of Cx46 with that of Cx32 was shown to reduce unitary conductance fivefold, convert open hemichannel current rectification from inward to outward and change permeability from cation- to anion-preferring (Trexler et al., 2000). The simplest interpretation of these results is that the E1 domain contributes to the pore and differences in sequence between Cx32 and Cx46 are responsible for the differences in open-channel properties. Fig. 1 A shows a sequence comparison of the E1 domains of Cx46, Cx32, and Cx43. Based on dye spread and ionic substitution studies in cell pairs, Cx46 exhibits a preference for cations, Cx32 for anions, and Cx43 no obvious preference on the basis of charge (Trexler et al., 2000). In Cx46, 5 residues with negatively charged side chains are clustered in a 10-residue stretch in E1 starting at E42 at the TM1/E1 border. In Cx32 and Cx43, two of these residues, E43 and D51, are replaced with Ser or Ala. Cx32 contains an additional positively charged Lys in place of Q49 giving Cx46 more negative charge in this region of E1 than Cx32 or Cx43. Because the combination of charges at positions 49 and 51 correlated with the selectivity characteristics of Cx46, Cx32, and Cx43, we modified the Cx46*32E1 chimera by converting residues 49 and 51 back to Cx46 sequence (Cx46*32E1(K49Q + S51D). The conductance of this channel was restored to ∼65% of wtCx46 (measured as the slope conductance at Vm = 0) and the open hemichannel I-V relation converted back to inwardly rectifying (Fig. 1 B). These results indicate that either or both residues at positions 49 and 51 in E1 of Cx46 are important for the effects caused by the Cx32 E1 substitution.


Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.

Kronengold J, Trexler EB, Bukauskas FF, Bargiello TA, Verselis VK - J. Gen. Physiol. (2003)

Residues in E1 of Cx46 near the TM1 border of Cx46 are important for the effects caused by the Cx46*32E1 substitution. (A) Sequence comparison of E1 domains of Cx46, Cx32, and Cx43. The accepted topology of a connexin subunit has four transmembrane (TM) domains, extracellular E1 and E2 loops, and cytoplasmic NH2-terminal (NT), loop (CL), and carboxy terminal (CT) domains. E42 resides at the TM1/E1 border and R76 at the E1/TM2 border. (B) Representative recordings of Cx46, Cx46*32E1, and Cx46*32E1(K49Q + S51D) hemichannel currents in Xenopus oocytes obtained with 8-s voltage ramps from −70 to +70 mV applied to cell-attached patches containing single hemichannels. The solid gray lines represent exponential fits to the open-state current. Substitution of the E1 domain of Cx32 into Cx46 (Cx46*32E1) substantially reduced unitary conductance and converted open-channel current rectification from inward to outward. Restoring the residues at positions 49 and 51 in the Cx46*32E1 chimera back to Cx46 sequence largely restored wild-type Cx46 properties.
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Related In: Results  -  Collection

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fig1: Residues in E1 of Cx46 near the TM1 border of Cx46 are important for the effects caused by the Cx46*32E1 substitution. (A) Sequence comparison of E1 domains of Cx46, Cx32, and Cx43. The accepted topology of a connexin subunit has four transmembrane (TM) domains, extracellular E1 and E2 loops, and cytoplasmic NH2-terminal (NT), loop (CL), and carboxy terminal (CT) domains. E42 resides at the TM1/E1 border and R76 at the E1/TM2 border. (B) Representative recordings of Cx46, Cx46*32E1, and Cx46*32E1(K49Q + S51D) hemichannel currents in Xenopus oocytes obtained with 8-s voltage ramps from −70 to +70 mV applied to cell-attached patches containing single hemichannels. The solid gray lines represent exponential fits to the open-state current. Substitution of the E1 domain of Cx32 into Cx46 (Cx46*32E1) substantially reduced unitary conductance and converted open-channel current rectification from inward to outward. Restoring the residues at positions 49 and 51 in the Cx46*32E1 chimera back to Cx46 sequence largely restored wild-type Cx46 properties.
Mentions: Substitution of the E1 domain of Cx46 with that of Cx32 was shown to reduce unitary conductance fivefold, convert open hemichannel current rectification from inward to outward and change permeability from cation- to anion-preferring (Trexler et al., 2000). The simplest interpretation of these results is that the E1 domain contributes to the pore and differences in sequence between Cx32 and Cx46 are responsible for the differences in open-channel properties. Fig. 1 A shows a sequence comparison of the E1 domains of Cx46, Cx32, and Cx43. Based on dye spread and ionic substitution studies in cell pairs, Cx46 exhibits a preference for cations, Cx32 for anions, and Cx43 no obvious preference on the basis of charge (Trexler et al., 2000). In Cx46, 5 residues with negatively charged side chains are clustered in a 10-residue stretch in E1 starting at E42 at the TM1/E1 border. In Cx32 and Cx43, two of these residues, E43 and D51, are replaced with Ser or Ala. Cx32 contains an additional positively charged Lys in place of Q49 giving Cx46 more negative charge in this region of E1 than Cx32 or Cx43. Because the combination of charges at positions 49 and 51 correlated with the selectivity characteristics of Cx46, Cx32, and Cx43, we modified the Cx46*32E1 chimera by converting residues 49 and 51 back to Cx46 sequence (Cx46*32E1(K49Q + S51D). The conductance of this channel was restored to ∼65% of wtCx46 (measured as the slope conductance at Vm = 0) and the open hemichannel I-V relation converted back to inwardly rectifying (Fig. 1 B). These results indicate that either or both residues at positions 49 and 51 in E1 of Cx46 are important for the effects caused by the Cx32 E1 substitution.

Bottom Line: Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified.The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits.If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA.

ABSTRACT
Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

Show MeSH
Related in: MedlinePlus