TY - JOUR
T1 - Neurons and β-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity
AU - Bukauskas, Feliksas F.
N1 - Funding Information:
We thank Dr. Garry E. Kiefer, chief executive officer of Macrocyclics (Dallas, TX), for the kind supply of EAM-1 and EAM-2 dyes; Dr. Klaus Willecke for kindly providing the constructs of Cx36; and PhD student Nerijus Paulauskas and Dr. Angele Bukauskiene for excellent technical assistance. This work was supported by National Institutes of Health grants HL084464 and NS072238 (to F. F. B.).
PY - 2012/6
Y1 - 2012/6
N2 - We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (Pγ), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P γ of Cx36 for cationic dyes (EAM-1+ and EAM-2 +) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350-). In addition, Pγ for Lucifer yellow (LY2-) is approximately fourfold smaller than that for AFl-350-, which suggests that the higher negativity of LY2- significantly reduces permeability. The Pγ of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the Pγs of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P γ of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K+, a major charge carrier in cell-cell communication.
AB - We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (Pγ), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P γ of Cx36 for cationic dyes (EAM-1+ and EAM-2 +) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350-). In addition, Pγ for Lucifer yellow (LY2-) is approximately fourfold smaller than that for AFl-350-, which suggests that the higher negativity of LY2- significantly reduces permeability. The Pγ of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the Pγs of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P γ of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K+, a major charge carrier in cell-cell communication.
KW - Cationic selectivity
KW - Connexin
KW - Gap junction
KW - Intercellular communication
KW - Permeability
KW - Voltage gating
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U2 - 10.1007/s00232-012-9445-3
DO - 10.1007/s00232-012-9445-3
M3 - Article
C2 - 22752717
AN - SCOPUS:84864356601
SN - 0022-2631
VL - 245
SP - 243
EP - 253
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
IS - 5-6
ER -