Regulation of connexin36 gap junction channels by n‐alkanols and arachidonic acid

Abstract

Key points• Under control conditions only one of ∼128 connexin (Cx)36 gap junction channels assembled in junctional plaques are open at a given time. This ratio was increased several times by short carbon chain n-alkanols and BSA, and reduced by long carbon chain n-alkanols.• Bovine serum albumin (BSA) increased junctional conductance (g j ) by removal of polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA) from the plasma membrane of HeLaCx36-EGFP cells. BSA modified with 1,2-cyclohexanedione, which does not bind PUFAs, did not affect g j .• A primary culture of pancreatic β-cells, expressing solely Cx36, shows similar properties as HeLa transfectants, i.e. g j increases under an exposure to BSA and hexanol, while decanol and nonanol caused full uncoupling.• Methyl arachidonyl fluorophosphonate (MAFP) and thapsigargin, inhibitor and activator of AA synthesis, increased and reduced g j , respectively.• The g j -enhancing effect of hexanol did not change during combined application with MAFP and BSA, whereas AA and thapsigargin reduced the potentiating effect of hexanol.Abstract We examined junctional conductance (g j ) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic β-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased g j , while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and g j increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and β-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase g j by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.

Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

“…Carbenoxolone, curiously, increased Cx36-mediated dye uptake, inhibited Cx30-mediated dye uptake, and displayed a relatively low potency toward Cx43, Px1, and P2X 7 . The increase in Cx36-mediated uptake may reflect relief from inhibition by intramembrane arachidonic acid as reported for short chain alcohols on Cx36 but not Cx43 gap junctional channels (86). Carbenoxolone has previously been reported to display a partial effect on Yo-Pro uptake in Px1-expressing HEK293 cells (87), whereas much higher potency is found in electrophysiological experiments in both oocytes and complex cell systems expressing Px1 (73,85).…”

The SH3-binding domain of Cx43 participates in loop/tail interactions critical for Cx43-hemichannel activity

“…Under normal (healthy) conditions, Cx43 gap junctions are open (this does not mean that all channels in a gap junction are open [30]). Under ischemic conditions or acidification, Cx43 gap junctions close due to an interaction of the loop with the tail, according to a particle receptor model.…”

Regulation of Connexin-Based Channels by Fatty Acids

“…PUFAs also regulated GJC activity in a transfected cell line. For example, AA inhibited GJCs in Cx36 transfected HeLa cells, and GJC activity was restored after washout (Marandykina et al, 2013). …”

“…Although it is not easy to characterize the effect of PUFAs on GJC activity, the effect of AA has been consistently associated to the same response: inhibition of GJC activity (Giaume et al, 1989; Fluri et al, 1990; Hii et al, 1995; Marandykina et al, 2013). But curiously, this type of response is absent in Xenopus oocytes, where AA does not affect GJCs formed by Cx46 (Retamal et al, 2011).…”