Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions

Abstract

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions.

Systems View of Deconditioning During Spaceflight Simulation in the PlanHab Project: The Departure of Urine 1 H-NMR Metabolomes From Healthy State in Young Males Subjected to Bedrest Inactivity and Hypoxia

“…The unique crossover design allowed us to include responses of the same participants to all three experimental variants, NBR, HBR, and HAmb, under the controlled nutritional, environmental, and experimental conditions. The same general trends of body deconditioning were recovered in this study based on 1 H-NMR metabolomics of urine, as described before using different sets of markers and approaches in the PlanHab subprojects ( Debevec et al, 2014a , 2016b ; Rittweger et al, 2016 ; Simpson et al, 2016 ; Strewe et al, 2017 , 2018 ; Stavrou et al, 2018a , b ; Supplementary Table 1 ). This shows large congruence between the various independently collected datasets within the PlanHab and the metabolomics approach used in this study.…”

“…The approach adopted in this study provides an opportunity to generate new hypotheses on metabolic pathway perturbation. One can indeed hypothesize that the metabolites involved in metabolic pathways identified in this study in fact act as signaling molecules [or account for lack of these (e.g., in HBR, NBR)] involved in the PlanHab symptoms as detailed in Figure 1 : insulin resistance, low-grade inflammation, different mitochondrial function, miRNA expression in large muscles, differences in lipid oxidation, mood changes, and depression ( Debevec et al, 2014a , 2016b ; Rittweger et al, 2016 ; Simpson et al, 2016 ; Sket et al, 2017a , b , 2018 ; Strewe et al, 2017 , 2018 ; Stavrou et al, 2018a , b ; Supplementary Table 1 ). In addition to those listed above, groups of metabolites identified in this study were also associated with: (i) the chronic obstructive pulmonary disease (COPD) ( Adamko et al, 2015 ; Za̧bek et al, 2015 ) and included metabolites such as 3-hydroxyisovalerate, 2-hydroxyisobutyrate, creatinine, formate, taurine, urea, choline, isoleucine, pantothenate, valine, and its degradation to beta-aminoisobutyric acid during metabolism of branched-chain amino acids suggest increased catabolism associated with COPD; (ii) cardiovascular disease as a results of associated chain of events such as tissue hypoxia (gut ischemia) due to reduced oxidative phosphorylation and energy production that lead to pulmonary hypertension, systemic inflammatory responses, and increased risk of cardiovascular disease, type 2 diabetes, depression, and osteoporosis ( Jones, 2014 ).…”

“…Acquisition of clinical, exercise, dietary, and ambiental data were described in detail before ( Debevec et al, 2014a , 2016a ; Simpson et al, 2016 ). The in-house database ( Sket et al, 2017b ) containing over 13,000 entries based on all measured variables in the PlanHab experiment (i.e., clinical, inflammation, immune, human physiology, and nutrition data next to the experimental design and characteristics of the participants) was checked for consistency and updated with recent publications related to the PlanHab project ( Debevec et al, 2014a , 2016b , 2018 ; Keramidas et al, 2016 ; Louwies et al, 2016 ; Rittweger et al, 2016 ; Rullman et al, 2016 , 2018 ; Simpson et al, 2016 ; Morrison et al, 2017 ; Strewe et al, 2017 ; Salvadego et al, 2018 ; Sarabon et al, 2018 ; Stavrou et al, 2018a , b ; Ciuha et al, 2020 ). The in-house database was used to identify parameters that differed significantly between the experimental variants over the course of the experiment as described before ( Sket et al, 2017b , 2018 ).…”

Connexins-Based Hemichannels/Channels and Their Relationship with Inflammation, Seizures and Epilepsy

“…These channels, through which cells are electrically and chemically coupled, can be formed from homomeric or heteromeric hemichannels, and are therefore called homotypic or heterotypic channels, respectively. The conductance of these Cxs-based channels and hemichannels is regulated by calcium concentration [13,14], intracellular pH [15,16,17,18], some neurotransmitters, such as serotonin and dopamine [19,20], the trans-junctional voltage (relative voltage difference between coupled cells), and the membrane voltage [21,22]. The conductance is related to the type of Cxs that make up the channels and hemichannels; for example, Cx36 has a conductance of 10–15 pS, while Cx45 and Cx43 have a conductance of 27.84 ± 0.25 pS [23,24,25] and 36.8 ± 0.54 pS [25], respectively.…”

The Role of Gap Junction in Regulating Vascular Function and Its Relationship with Hypertension

“…The regulation of gap junction function is significant for cell communication. Cytoplasmic pH, Ca 2+ , voltage, oncogenes, nucleotides, hormones, neurotransmitters, lipids, growth factors, and many exogenous chemicals can regulate function of gap junction by different levels including transcription, translation and post-translation modification, which is achieved by a variety of mechanisms [11][12][13][14].…”