State-Dependent cAMP Binding to Functioning HCN Channels Studied by Patch-Clamp Fluorometry

Authors

Shengjun Wu, Virginia Commonwealth University
Zhanna V. Vysotskaya, Virginia Commonwealth University
Xinping Xu, Virginia Commonwealth University
Changan Xie, Virginia Commonwealth University
Qinglian Liu, Virginia Commonwealth University
Lei Zhou, Virginia Commonwealth UniversityFollow

Document Type

Article

Original Publication Date

2011

Journal/Book/Conference Title

The Biophysical Journal

Volume

100

Issue

5

First Page

1226

Last Page

1232

DOI of Original Publication

10.1016/j.bpj.2011.01.034

Comments

Originally published at http://dx.doi.org/10.1016/j.bpj.2011.01.034

Under an Elsevier user license

Date of Submission

February 2015

Abstract

Abstract

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors.

Rights

From The Biophysical Journal, Wu, S., Vysotskaya, Z.V., Xu, X., et al., State-Dependent cAMP Binding to Functioning HCN Channels Studied by Patch-Clamp Fluorometry, Vol. 100, Page 1226. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. Reprinted with permission.

Is Part Of

VCU Physiology and Biophysics Publications