Low and High pH Gating of Connexin43 Channels

Abstract

Intracellular pH (pHi) regulation in myocardial tissue is crucial to preserving cardiac function. H+-ions can flow passively at high rates through connexin gap-junctions to neighbouring cells, helping to maintain pHi-uniformity in multicellular tissue. In ventricular myocytes, this form of spatial H+-dissipation is regulated by pHi at the junction.We have studied the response of connexin43, the main ventricular junctional channel-isoform, to high and low pHi. Transjunctional conductance (Gj) was measured by double whole-cell voltage clamp in HeLa or N2A cell-pairs transfected with connexin43. pHi was manipulated with 80mM acetate or 20mM trimethylamine in the superfusate. Acid (<6.7) and alkaline (∼7.5) pHi reversibly reduced (<5min) cell-to-cell Gj by 35±4% and 56±3% respectively (n=4-5), relative to the values at resting pHi (∼7.0). Acute electrical uncoupling at high and low pHi occurred with no change in single channel conductance (2mM halothane; 122±pS, n=250), suggesting a channel-gating mechanism. Cells expressing a truncated C-terminus mutant of connexin43, showed no conductance response to pHi-changes.Using a different approach, we measured apparent junctional H+-permeability (PHapp) through connexin43. One cell of a pair was acid-loaded by photolytically uncaging H+ from an intracellular donor compound (nitrobenzaldehyde), while pHi was confocally imaged in both cells (SNARF-1). Cell-to-cell H+-diffusion was inhibited by β-glycerrhetinic acid (connexin-blocker, 60μM) and was negligible in wild type cell-pairs. In HCO3−-free buffer, presetting pHi to 6.6 or 7.3, reduced PHapp by 81±6% and 76±14%, respectively (n=5-19). PHapp returned to initial values when pHi was allowed to recover. The presence of 5%CO2/22mMHCO3− in the perfusate doubled PHapp.In conclusion, connexin43 gap-junctions exhibit reversible block at both acid and alkaline pHi, possibly by a gating mechanism involving the cytoplasmic C-terminal tail. This suggests a key role for connexin43-channels in coordinating cell-to-cell electrical coupling and spatial pHi regulation during metabolic stress.