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Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes
G Droogmans1, J Prenen, J Eggermont
1Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.
Abstract:
We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 +/- 2.1 pS (n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 microM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.
Insights
Calix[4]arenes inhibit volume-regulated anion channels (VRAC) by blocking the pore at positive potentials. This open-channel block is influenced by pH and voltage, suggesting protonation affects calixarene binding within the VRAC pore.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Physiology
Background:
- Volume-regulated anion channels (VRAC) are crucial for cell volume regulation and cellular signaling.
- Calix[4]arenes are macrocyclic compounds with potential applications in modulating ion channel function.
Purpose of the Study:
- To investigate the effects of TS- and TS-TM-calix[4]arenes on VRAC currents.
- To elucidate the mechanism of calixarene-channel interaction and voltage/pH dependence.
Main Methods:
- Patch-clamp electrophysiology on cultured calf pulmonary artery endothelial cells.
- Analysis of current inhibition, noise, and single-channel conductance.
- Development of a kinetic model to describe calixarene binding to VRAC.
Main Results:
- TS- and TS-TM-calix[4]arenes caused voltage-dependent inhibition of VRAC currents, with maximal block at 30-50 mV.
- Lowering extracellular pH enhanced the block and shifted the potential of maximum inhibition.
- Calixarene-induced current noise analysis yielded a single-channel conductance of 9.3 ± 2.1 pS.
- A model indicated calixarene binding to a site within the VRAC pore, occluding the channel at positive potentials.
Conclusions:
- Calix[4]arenes induce an open-channel block of VRAC.
- The block is voltage-dependent and influenced by the protonation state of the binding site within the channel.
- Calixarenes can permeate the VRAC at more positive potentials after binding.