Rational design of new NO and redox sensitivity into connexin26 hemichannels

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.

Altered CO2 sensitivity of connexin26 mutant hemichannels in vitro

“…Given that we had no prior knowledge or hypothesis as to what the mutations should do to CO 2 sensitivity, and that we have seen all possible outcomes with respect to effects on CO 2 sensitivity for the various mutations, we believe our assay to be unbiased. In our prior studies, the dye loading results obtained using the methods outlined here correspond very closely to electrophysiological measurements of hemichannel activity (Huckstepp et al 2010a;Meigh et al 2013Meigh et al , 2015, giving us further reassurance that our measurements were unbiased.…”

“…; Meigh et al. , , ). The parental HeLa cells do not load with dye in response to either a CO 2 stimulus or removal of extracellular Ca 2+ following transfection with an empty vector (Meigh et al.…”

“…The parental HeLa cells do not load with dye in response to either a CO 2 stimulus or removal of extracellular Ca 2+ following transfection with an empty vector (Meigh et al. , , ). The parental HeLa cells thus have no endogenous hemichannels or CO 2 sensitivity and are an ideal expression system for Cx26 and its mutant subunits.…”

“…We evaluated the CO 2 sensitivity of wild type and mutant Cx26 hemichannels via our previously established and validated dye-loading assay (Huckstepp et al 2010a;Meigh et al 2013Meigh et al , 2014Meigh et al , 2015. The parental HeLa cells do not load with dye in response to either a CO 2 stimulus or removal of extracellular Ca 2+ following transfection with an empty vector (Meigh et al 2013(Meigh et al , 2014(Meigh et al , 2015. The parental HeLa cells thus have no endogenous hemichannels or CO 2 sensitivity and are an ideal expression system for Cx26 and its mutant subunits.…”

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

“…We used a dye loading protocol that has been developed and extensively described in our prior work (Dospinescu et al, 2019;Huckstepp et al, 2010a;Meigh et al, 2015;Meigh et al, 2013;Meigh et al, 2014). HeLa-DH cells were plated onto coverslips and transfected with Cx26 DN .…”