Inhibition of an outwardly rectifying anion channel by HEPES and related buffers |
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Authors: | J W Hanrahan J A Tabcharani |
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Institution: | (1) Department of Physiology, McGill University, H3G 1Y6 Montreal, Quebec, Canada |
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Abstract: | Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies 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 andbis-Tris at millimolar concentrations. HEPES (pKa=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES
i
) and this effect increased with HEPES
i
] so that conductance at the reversal potential was diminished 25% with 10mm HEPES
i
)and 70% at very high HEPES
i
]. HEPES
i
block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES
i
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 (pKa=7.54), substituted glycine buffers (pKa=8.1–8.4) andbis-Tris (pKa=6.46) had no measurable effect on conductance and appear suitable for use with this channel. |
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Keywords: | epithelia chloride channel cystic fibrosis PANC-1 TES |
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