Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32

Matthew J. Brennan, Jennifer Karcz, Nicholas R. Vaughn, Yvonne Woolwine-Cunningham, Adam D. DePriest, Yerko Escalona, Tomas Perez-Acle, I. Martha Skerrett

Resultado de la investigación: Article

2 Citas (Scopus)

Resumen

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, porefacing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

Idioma originalEnglish
Páginas (desde-hasta)17074-17084
Número de páginas11
PublicaciónJournal of Biological Chemistry
Volumen290
N.º28
DOI
EstadoPublished - 10 jul 2015

Huella dactilar

Connexins
Gap Junctions
Tryptophan
Substitution reactions
Scanning
Electric potential
Facings
Connexin 43
Clamping devices
Xenopus
Set theory
Oocytes
Electrodes
Membranes
Lipids
Water

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)
  • Molecular Biology
  • Cell Biology

Citar esto

Brennan, M. J., Karcz, J., Vaughn, N. R., Woolwine-Cunningham, Y., DePriest, A. D., Escalona, Y., ... Skerrett, I. M. (2015). Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32. Journal of Biological Chemistry, 290(28), 17074-17084. https://doi.org/10.1074/jbc.M115.650747
Brennan, Matthew J. ; Karcz, Jennifer ; Vaughn, Nicholas R. ; Woolwine-Cunningham, Yvonne ; DePriest, Adam D. ; Escalona, Yerko ; Perez-Acle, Tomas ; Skerrett, I. Martha. / Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32. En: Journal of Biological Chemistry. 2015 ; Vol. 290, N.º 28. pp. 17074-17084.
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title = "Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32",
abstract = "Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, porefacing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.",
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Brennan, MJ, Karcz, J, Vaughn, NR, Woolwine-Cunningham, Y, DePriest, AD, Escalona, Y, Perez-Acle, T & Skerrett, IM 2015, 'Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32', Journal of Biological Chemistry, vol. 290, n.º 28, pp. 17074-17084. https://doi.org/10.1074/jbc.M115.650747

Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32. / Brennan, Matthew J.; Karcz, Jennifer; Vaughn, Nicholas R.; Woolwine-Cunningham, Yvonne; DePriest, Adam D.; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I. Martha.

En: Journal of Biological Chemistry, Vol. 290, N.º 28, 10.07.2015, p. 17074-17084.

Resultado de la investigación: Article

TY - JOUR

T1 - Tryptophan scanning reveals dense packing of connexin transmembrane domains in gap junction channels composed of connexin32

AU - Brennan, Matthew J.

AU - Karcz, Jennifer

AU - Vaughn, Nicholas R.

AU - Woolwine-Cunningham, Yvonne

AU - DePriest, Adam D.

AU - Escalona, Yerko

AU - Perez-Acle, Tomas

AU - Skerrett, I. Martha

PY - 2015/7/10

Y1 - 2015/7/10

N2 - Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, porefacing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

AB - Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, porefacing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

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