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Cosegregation of permeability and single-channel conductance in chimeric connexins.

ABSTRACT: The physiological function of gap junction channels goes well beyond their initially discovered role in electrical synchronization of excitable cells. In most tissues, gap junction cells facilitate the exchange of second messengers and metabolites between cells. To test which parts of the channels formed by connexins determine the exclusion limit for the transit of molecules in the size range of second messengers and metabolites a domain exchange approach was used in combination with an accessibility assay for nonelectrolytes and flux measurements. The experimental results suggest that two open hemichannel forming connexins, Cx46 and Cx32E(1)43, differ in accessibility and permeability. Sucrose is at the exclusion limit for Cx46 channels whereas sorbitol is at the exclusion limit for Cx32E(1)43 channels. In chimeras between these connexins, where the first transmembrane segment M1 is exchanged, the exclusion limits correlate with those of the M1 donor. The same segregation was found in a separate study for the unitary conductance of the channels. Thus, conductance and permeability/accessibility of the channels cosegregate with M1.

SUBMITTER: Ma M

PROVIDER: S-EPMC1367014 | biostudies-other | 2006 Jan

REPOSITORIES: biostudies-other

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Cosegregation of permeability and single-channel conductance in chimeric connexins.

Ma Meiyun M Dahl Gerhard G

Biophysical journal 20051007 1

The physiological function of gap junction channels goes well beyond their initially discovered role in electrical synchronization of excitable cells. In most tissues, gap junction cells facilitate the exchange of second messengers and metabolites between cells. To test which parts of the channels formed by connexins determine the exclusion limit for the transit of molecules in the size range of second messengers and metabolites a domain exchange approach was used in combination with an accessib ...[more]

PMID: 16214854

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Project description:The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

Dataset Information

Cosegregation of permeability and single-channel conductance in chimeric connexins.

Publications

Cosegregation of permeability and single-channel conductance in chimeric connexins.

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