Structural determinants underlying permeant discrimination of the Cx43 hemichannel
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Structural determinants underlying permeant discrimination of the Cx43 hemichannel. / Nielsen, Brian Skriver; Zonta, Francesco; Farkas, Thomas; Litman, Thomas; Nielsen, Morten Schak; Macaulay, Nanna.
In: Journal of Biological Chemistry, Vol. 294, No. 45, 2019, p. 16789-16803.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Structural determinants underlying permeant discrimination of the Cx43 hemichannel
AU - Nielsen, Brian Skriver
AU - Zonta, Francesco
AU - Farkas, Thomas
AU - Litman, Thomas
AU - Nielsen, Morten Schak
AU - Macaulay, Nanna
PY - 2019
Y1 - 2019
N2 - Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichanne
AB - Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichanne
KW - connexin
KW - permeability
KW - pharmacology
KW - biophysics
KW - computer modeling
KW - connexon (hemichannel)
KW - gap junction
KW - connexin 30
KW - connexin 43
KW - ion conductivity
KW - membrane channel
U2 - 10.1074/jbc.RA119.007732
DO - 10.1074/jbc.RA119.007732
M3 - Journal article
C2 - 31554662
VL - 294
SP - 16789
EP - 16803
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 45
ER -
ID: 232483166