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08:33
Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
Published on: March 11, 2021
C-terminal movement during gating in cyclic nucleotide-modulated channels.
Kimberley B Craven1, Nelson B Olivier, William N Zagotta
1Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
The Journal of Biological Chemistry
|March 28, 2008
Summary
The study reveals that the SB triad in cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels forms in the closed state and expands upon opening. Manipulating this triad influences channel gating dynamics.
Area of Science:
- Molecular and Cellular Biology
- Biophysics
- Ion Channel Physiology
Background:
- Cyclic nucleotide-modulated channels, including CNG and HCN channels, are crucial for cellular signaling.
- Ligand binding induces conformational changes in the C-terminal regions, regulating channel activity.
- The C-terminal intersubunit interface contains a conserved 'SB triad' of charged residues involved in channel gating.
Purpose of the Study:
- To test the hypothesis that the SB triad forms in the closed state and breaks upon channel opening.
- To investigate the structural and functional consequences of manipulating the SB triad in CNGA1 and HCN2 channels.
Main Methods:
- Site-directed mutagenesis and application of charged reagents to alter SB triad residues in functioning CNGA1 channels.
- X-ray crystallography to determine the structure of the HCN2 C-terminal region with an E462R mutation.
- Cysteine cross-linking to restrict movement within the SB triad region.
Main Results:
- Charge reversal mutations in the SB triad increased the favorability of channel opening.
- Crystal structure of the mutated HCN2 channel showed a rearranged SB triad, facilitating easier opening.
- Cross-linking that prevented SB triad movement inhibited channel opening, while longer cross-linkers did not.
Conclusions:
- The SB triad is integral to the closed state of CNG and HCN channels.
- Expansion of the SB triad region is a key event during channel opening.
- These findings elucidate a novel gating mechanism for cyclic nucleotide-modulated ion channels.
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