Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties

A. P. Moreno, B. Eghbali, David C. Spray

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Abstract

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (g(j)) to transjunctional voltages (V(j)s) of either polarity, with the ratio of minimal to maximal g(j) (g(min)/g(max)) being ~0.1 at the highest V(j)s. Steady-state g(j) values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which g(j) was reduced by 50% was ~25-30 mV; A, the Boltzmann parameter describing voltage dependence, was ~0.06 (corresponding to an energy difference between states of ~1 kCal/mol and to ~2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (π > 5 s at 20-40 mV, π = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (α) was ~30% lower than that of the closing rate constant (β) over the V(j) range 0-70 mV; at higher voltages, voltage sensitivity of α and β saturated. The kinetic response of g(j) to a paradigm in which g(j) was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.

Original languageEnglish (US)
Pages (from-to)1267-1277
Number of pages11
JournalBiophysical Journal
Volume60
Issue number5
StatePublished - 1991

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Gap Junctions
Connexins
Patch-Clamp Techniques
Cell Communication
Electrodes
Plasmids
Complementary DNA
Cell Line
Liver

ASJC Scopus subject areas

  • Biophysics

Cite this

Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties. / Moreno, A. P.; Eghbali, B.; Spray, David C.

In: Biophysical Journal, Vol. 60, No. 5, 1991, p. 1267-1277.

Research output: Contribution to journalArticle

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