The Effect of Arachidonic Acid on Junctional Conductance and Gating of Connexin 36 Gap Junction Channels and their Modulation by N-Alkanols

Abstract

Arachidonic acid (AA) is one of major components of membrane phospholipids and is actively involved in the regulation of junctional conductance (gj). N-alkanols are well known inhibitors of gap junction (GJ) channels and have been shown to reduce neurological tremors. In this study, we examined function of Cx36 GJ channels, which are expressed in neurons and β-cells of pancreas, under control conditions and application of factors modulating concentration of AA in the plasma membrane. We found that in HeLa cells expressing Cx36-EGFP, short carbon chain alkanols (SCCAs), such as pentanol, hexanol and heptanol, increased gj by ∼3-fold. Conversely, long carbon chain alkanols (LCCAs), such as octanol, nonanol and decanol, uncoupled cells fully. We demonstrate that under control conditions only ∼0.003 of Cx36 GJ channels assembled in junctional plaques are functional, and this fraction increases by SCCAs and fatty acid free bovine serum albumin (BSA). BSA increased gj by 1.6-fold with EC50 of 2.3 M, while its modified form 1,2-cyclohexanedione (BSA-CHD), which does not bind AA, was ineffective. Voltage sensitive gating of Cx36 GJs was reduced by SCCA and BSA that explain in part their gj-enhancing effect. The inhibition of Cx36 GJ channels by AA can be rescued by BSA but not by BSA-CHD. MAFP and thapsigargin, inhibitor and activator of AA synthesis via phospholipase A2, increased and reduced gj, respectively. We assume that endogenous AA is one of key factors leading to low functional efficacy of Cx36 GJ channels under control conditions. Furthermore, we suggest that gj-enhancing effect of BSA and MAFP may be related with reduction of AA levels, while SCCAs limit AA's accessibility to its binding site on Cx36.