Rational design of new NO and redox sensitivity into connexin26 hemichannels.

Louise Meigh,Nicholas Dale,Jie Zhang,Daniel Cook

doi:10.1098/rsob.140208

Abstract

CO2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO2−, hemichannels comprising Cx26K125C can be opened by NO2− and NO donors. However, NO2− was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO2−. In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

Highlights

The human genome contains 21 connexin genes [1]
We examined the gating of Cx26 hemichannels via an established assay, whereby loading of CBF into Cx26-expressing HeLa cells is a measure of hemichannel opening [5]
We found that 100 mM nitrite caused dye loading in Cx26K125Cexpressing HeLa cells ( p 1⁄4 0.008 compared with background control, figure 3a,b,d)

Summary

Introduction

The human genome contains 21 connexin genes [1]. Connexins are one of the main gene families that can form gap junctions. The hemichannels dock to form a continuous passageway linking the cytoplasm of the coupled cells that permits transfer of ions and small molecules. In addition to this canonical function of connexins as gap junctions, undocked hemichannels can have physiological functions [2,3]. The carbamylated residue can form a salt bridge to the neighbouring subunit in the hexamer [5]. This opens the hemichannel, and allows release of ATP, which is an important biological signalling molecule [6]. The CO2-dependent opening of Cx26, and consequent release of ATP, is a key mechanism in the regulation of breathing by CO2 [6]

Methods

Results

Discussion

Conclusion