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Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

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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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  1. Department of Physiology, McGill University, H3G 1Y6, Montreal, Quebec, Canada

    J. W. Hanrahan & J. A. Tabcharani

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  1. J. W. Hanrahan
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  2. J. A. Tabcharani
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Hanrahan, J.W., Tabcharani, J.A. Inhibition of an outwardly rectifying anion channel by HEPES and related buffers. J. Membrain Biol. 116, 65–77 (1990). https://doi.org/10.1007/BF01871673

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  • DOI: https://doi.org/10.1007/BF01871673

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