Inhibition of an outwardly rectifying anion channel by HEPES and related buffers.
The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studied using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris and bis-Tris at millimolar concentrations. HEPES (pK alpha = 7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6 mM HEPES on the cytoplasmic side (HEPESi) and this effect increased with [HEPESi] so that conductance at the reversal potential was diminished approximately 25% with 10 mM HEPEsi and approximately 70% at very high [HEPESi]. HEPESi block was relieved by applying positive voltage but positive currents were not consistent with a Woodhull-type blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPESi concentration at constant [HEPES-] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK alpha = 7.54), substituted glycine buffers (pK alpha = 8.1-8.4) and bis-Tris (pK alpha = 6.46) had no measurable effect on conductance and appear suitable for use with this channel.
Hanrahan
J W
JW
Department of Physiology, McGill University, Montreal, Quebec, Canada.
Tabcharani
J A
JA
D002021:Buffers; D002460:Cell Line; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D004553:Electric Conductivity; D006531:HEPES / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D015394:Molecular Structure; D010879:Piperazines / Q000494:pharmacology*
D002021:Buffers; D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D010879:Piperazines; D006531:HEPES