Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics

Vytautas Raškevičius,Visvaldas Kairys,Vytenis Arvydas Skeberdis,Lina Rimkutė,Alina Marandykina,Vaidas Jotautis,Mintautė Kazokaitė

doi:10.1042/bsr20171323

Vytautas Raškevičius, Visvaldas Kairys + Show 5 more

Open Access

https://doi.org/10.1042/bsr20171323

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Abstract

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

Highlights

Gap junction (GJ) channels (Figure 1A,B) are composed of two apposed hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication
Alcohols and anesthetics evoke their effects by binding to the specific residues in the transmembrane domain that lines the ion channel pore [13,38], and it is proposed that pentameric ligand-gated ion channel (pLGIC) contain intrasubunit-inhibitory and inter-subunit-potentiating sites [16,33]
GJs or, in other words, electrical synapses in the CNS can be inhibited by general anesthetics suggesting that they may play a role in anesthesia [19]

Summary

Introduction

Gap junction (GJ) channels (Figure 1A,B) are composed of two apposed hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. We have demonstrated that Cx36 distinguishes from other Cxs by its opposite response to widely used GJ uncouplers, such as n-alcohols or general anesthetics While these agents inhibit Cx45 GJs [40], they strongly potentiate Cx36 GJs [28]. We hypothesized that stimulatory and inhibitory effects of n-alcohols may originate from their interaction with different domains of Cx36 c 2018 The Author(s) We demonstrate by dual whole-cell patch-clamp experiments that these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical to other Cxs that lack respective cysteines or/and a specific docking pocket for these compounds

Materials and methods

Results

Discussion