Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels.

Martina Rangl,Atsushi Miyagi,Henning Stahlberg,Simon Scheuring,Crina M Nimigean,Julia Kowal

doi:10.1038/ncomms12789

Abstract

Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

Highlights

Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain
The structure and dimensions were in agreement with results obtained by conventional AFM15 and earlier work[13]; the fast HS-atomic force microscopy (AFM) imaging acquisition in the sub-second range revealed that about 10% of the molecules dynamically switched between two conformations of different heights
We captured the dynamics of the conformational changes of MloK1 channels upon cAMP unbinding using high-speed AFM (HS-AFM)

Summary

Introduction

Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. We image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. Cyclic nucleotide-modulated (CNM) ion channels are key players throughout the entire nervous system as they regulate certain modes of signal transduction[3,4] and play a role in neuronal excitability in brain and pacemaking in heart cells[5,6] They detect levels of intracellular cyclic AMP or GMP (cAMP or cGMP) by direct binding to a specialized cyclic nucleotide-binding domain (CNBD). This chemical information, that is, ligand binding, is translated into an electrical response by opening of the channel pore allowing ion flow across the membrane. We propose that upon ligand binding the channel switches from a set of flexible conformations created by the highly dynamic unliganded CNBDs to an ordered conformation with stable, liganded CNBDs, and that these conformations are important for gating

Methods

Results

Conclusion