Biophysics 204 lectures - Ion Channels
Dan Minor, Ph.D., CVRB 452Z, daniel.minor@ucsf.edu
11, 13, 15, & 18 February 13
The format for these lectures will be the following:
11, 13, 15 February 13– Minor will lecture on channels to set the foundation for the discussion
session, which will be a focus session.
If you want to read an up-to-date perspective of at least part of the field and the historical context
see:
Bezanilla Neuron 60:456-468 (2008)
18 February 13 –– Channel desensitization – Glutamate Receptors:
1) Sun et al. ‘Mechanism of glutamate receptor desensitization’, Nature 417:245-253 (2002)
2) Armstrong et al. ‘Measurement of conformational changes accompanying desensitization in an
ionotropic glutamate receptor’, Cell 127:85-97 (2006)
3) Weston et al. ‘Conformational restriction blocks glutamate receptor desensitization’, Nature
Structural and Molecular Biology 13:1120-1127 (2006)
For background see:
Mayer, ML, ‘Emerging models of glutamate receptor ion channel structure and function’ Structure
10:1370-1380 (2011)
I have presented discussion questions here. Everyone should be prepared to discuss any
figure from any of the assigned papers and be prepared to present/lead the discussion
around the following questions.
The questions that I present here are provided to encourage discussion including
background and detailed resolution about the point if appropriate.
The best answers are not single word, but are thoughtful ones.
10 February 2012: Desensitization (or inactivation) is an important property of many ion channels.
The objective of these papers is to understand in structural terms how such a process might
happen.
Focus papers:
1) Sun et al. ‘Mechanism of glutamate receptor desensitization’, Nature 417:245-253 (2002)
2) Armstrong et al. ‘Measurement of conformational changes accompanying desensitization in an
ionotropic glutamate receptor’, Cell 127:85-97 (2006)
3) Weston et al. ‘Conformational restriction blocks glutamate receptor desensitization’, Nature
Structural and Molecular Biology 13:1120-1127 (2006)
4) Sobolevsky et al. X-ray structure, symmetry, and mechanism of an AMPA-subtype glutamate
receptor Nature 462:745-75456 (2009)
For background see:
Mayer, ML, ‘Glutamate receptors at atomic resolution’ Nature 440:456-462 (2006)
Assignments for discussion points
Paper 1- Introduction to the problem – Explain the difference between desensitization and
deactivation.
What are the functional consequences of the L483Y mutation and CTZ (Figure 1)?
What needs to be done to make Glutamate Receptor domains for crystallization?
What is the implication of the change dimer affinity by 10 5 caused by L483Y in the context of a
1,800Å2 interface? Does this influence your picture of protein-protein interactions?
How does CTZ bind? Are there differences with L483Y?
Explain Figure 3 and how the free energies are calculated.
Why do the authors think that L483Y and S754D mutants act independently?
Discuss the structural consequences of the S754D mutant.
Discuss the model for receptor function and the evidence.
What types of experiments would you propose to do next?
Paper 2Describe the modifications made to the receptor. Why did they need to engineer the changes?
How do they know it does not change the properties of the channel?
Explain the experiment and results in Figure 1. What do the authors learn about the resting and
desensitized conformation?
What is the rationale behind the experiment shown in Figure 2? What do the data indicate?
What is the caveat to the experiment?
Explain the significance of the S729C mutant and the experiments in Figures 3 and 4.
What is learned from the antagonism between The L483Y mutation and the S729C mutation?
Explain the crystallographic results in Figure 6 and discuss the model in Figure 7.
Paper 3 – Introduction – Background on AMPA vs. Kainate receptors
What is the rationale for the first engineering attempt to make non-desensitizing Kainate
receptors?
Figure 2 – Please explain each of the panels of the figure.
Did the first engineering attempt work?
What do you think the results from these experiments tell us?
How did the authors design the disulfide bond mutants?
Why are covalent dimers made at pH 8.0 but not pH 5.0?
What is the difference in the AUC analyses between Figures 2c,d and 3c,d?
Explain the Figure 5.
What does the structural comparison of the ELKQ mutant and disulfide dimer show? What is
the significance of the RMSD differences?
What does the cyclothiazide experiment (Figure 6c) suggest?
Summarize the findings with criticism and suggestions for future work and relate these findings to
those in Paper 2.
Paper 4– General discussion about context, advances, changes in interpretations