Connexin32 gap junction channels in stably transfected cells: Unitary conductance

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

Research output: Contribution to journalArticle

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Abstract

Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10pS). Macroscopic junctional conductance (g(j)) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (γ(j)) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic g(j) was > 1 nS; g(j) increased with time after dissociation (from 1.8 ± 0.6 [mean ± SE; n = 7] nS at 2 h after plating to 9.3 ± 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing g(j) by exposure to halothane or heptanol. Histograms of γ(j) values in transfected cells, in 10 experiments where > 100 transitions were measurable, displayed two peaks: 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.

Original languageEnglish (US)
Pages (from-to)1254-1266
Number of pages13
JournalBiophysical Journal
Volume60
Issue number5
StatePublished - 1991

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Gap Junctions
Halothane
Uncoupling Agents
Heptanol
Connexins
Patch-Clamp Techniques
Noise
Hepatocellular Carcinoma
Electrodes
Plasmids
Complementary DNA
Cell Line
Membranes
Liver

ASJC Scopus subject areas

  • Biophysics

Cite this

Connexin32 gap junction channels in stably transfected cells : Unitary conductance. / Moreno, A. P.; Eghbali, B.; Spray, David C.

In: Biophysical Journal, Vol. 60, No. 5, 1991, p. 1254-1266.

Research output: Contribution to journalArticle

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