Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina (original) (raw)

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to ∼400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is ∼90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH i from 7.2 to 7.8 increased gap junctional conductance (g j ) of ∼100-fold with pK a = 7.41. We show that this g j increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca 2+ ] o = ∼0.3 mm or below, demonstrating that a moderate reduction of [Ca 2+ ] o can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH i with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina. Abbreviations A h , coefficient characterizing the steepness of g j decay of AHC over V j ; AHC, apposed hemichannel; AF 350 , Alexa Fluor-350; F JP , fluorescence per unit area in en face JPs; F γ , fluorescence produced by a single GJ channel; F T , total fluorescence of JP; γ h,o , unitary conductance of AHC at the fully open state; γ h,res , unitary conductance of AHC at the residual state; γ o , unitary conductance of GJ channel at the fully open state; γ res , single channel conductance at the residual state; JP, junctional plaque; g j , transjunctional conductance; GJ, gap junction; AHC R and AHC L , rightand left-side AHCs; K , fraction of operating channels at any given time; MKR, modified Krebs-Ringer solution; N T , total number of physical channels in a JP; N f , number of functional channels; O-O, both AHCs are in the open state; C-C, both AHCs are in the closed state; O-C and C-O, one AHC is in the open state, while the other AHC is in the closed state; P j , junctional permeability; P γ , single channel permeability; ROI, region of interest; P o-o , P o-c , P c-o and P c-c , probabilities of channels to dwell in O-O, O-C, C-O and C-C states, respectively; UHC, unapposed hemichannel; V h,o , V j at which equilibrium constant between states of AHCs are equal to 1; V h , voltage across AHC; V j , transjunctional voltage.