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Patch-clamp fluorometry-based channel counting to determine HCN channel conductance.

ABSTRACT: Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide-regulated (HCN) channels. We use a well-characterized and homologous cyclic nucleotide-gated (CNG) channel to establish the relationship between macroscopic fluorescence intensity and the total number of channels. Subsequently, based on our estimate of the total number of HCN channels, we determine the single-channel conductance of HCN1 and HCN2 to be 0.46 and 1.71 pS, respectively. Such a small conductance would present a technical challenge for traditional electrophysiology. This PCF-based technique provides an alternative method for counting particles on cell membranes, which could be applied to biophysical studies of other membrane proteins.

SUBMITTER: Liu C

PROVIDER: S-EPMC4924933 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Patch-clamp fluorometry-based channel counting to determine HCN channel conductance.

Liu Chang C Xie Changan C Grant Khade K Su Zhuocheng Z Gao Weihua W Liu Qinglian Q Zhou Lei L

The Journal of general physiology 20160701 1

Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cycli ...[more]

PMID: 27353446

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Project description:BackgroundThere is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds.Methodology/principal findingsA comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0.Conclusions/significanceThe correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC(50); therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.

Dataset Information

Patch-clamp fluorometry-based channel counting to determine HCN channel conductance.

Publications

Patch-clamp fluorometry-based channel counting to determine HCN channel conductance.

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