Our research program over the past three decades has been guided by the fundamental tenet that voltage- gated channel proteins are modular in design and that the coupling between the component modules underlies their exquisite sensitivity to voltage and governs their functional diversity. For the new funding period, the thre main goals focus on: 1. The determination of the crystal structure of the channel in an open conformation attempting to understand the role of the sensor module. We will pursue the structure-function program of the pore module by supplementing the experimental conditions used to crystallize it with novel open-conformation stabilizers aiming to decode the open channel structure. 2. The elucidation of the origin of the voltage dependence in terms of the inherent properties of the sensorless pore, and on the regulation conferred by partner modules including a voltage sensor or a mechanical stress sensor. Numerous mutants generated at key locations on the PM structure favor the conductive state of the pore;accordingly, we will attempt to crystallize the most interesting candidates aiming to uncover an open structure. Having established the channel properties of the purified mechanosensitive Piezo proteins reconstituted in lipid bilayers leads us to apply our molecular dissection strategy to identify the structural determinants of the pore module and of the force sensor module. A vast number of chimeras, truncated constructs, and carefully selected mutants will be generated and functionally characterized along the lines previously reported. The ultimate aim is to confer mechanosensitivity to the robust PM scaffold by appending the force sensor module of Piezo proteins. 3. The structural and functional characterization of a newly discovered voltage-activated anion-selective channel (Xv). An immediate goal entails an in-depth functional characterization in terms of selectivity, permeation, and block of the purified full-length Xv and f its sensorless-PM after reconstitution in lipid bilayers. Attention will be directed to establish te voltage-dependence of activation given that Xv may represent a hyperpolarization-activated channel. An urgent task is to crystallize Xv and its PM using the lipid cubic phase approach we successfully applied for KvLm. The procedures we developed over this +30-year interval to investigate structure-function relationships together with newly acquired capability for structure determination augur a realistic, innovative, and productive continuation of our long-term commitment to a mechanistic understanding of voltage sensing in terms of the modular design of voltage-gated channel proteins.

Public Health Relevance

. Thanks again, Mauricio Mauricio Montal MD, PhD Distinguished Professor of Biological Sciences Section of Neurobiology University of California San Diego 9500 Gilman Drive La Jolla, CA 92093-0366 Tel: +1 858 534 0931 Fax: +1 858 822 3763 From: Grubelich, Claudette (NIH/NIGMS) [E] [GrubeliC@mail.nih.gov] Sent: Thursday, May 15, 2014 7:05 AM To: Montal, M. Cc: Jackson, Derek;Nie, Zhongzhen (NIH/NIGMS) [E] Subject: FW: Revised Specific Aims for Grant 2RO1 GM49711, PI: Mauricio Montal Dr. Montal, Please address the Modified Public Health Relevance Section with no more than 2 or 3 sentences, describe the relevance of this research to public health. Thank you. Claudette Grubelich GAB/NIGMS National Institutes of Health 45 Center Dr., Rm. 2AN24B Bethesda, MD 20892-6200 301-594-9251 From: Montal, M. [mailto:mmontal@ucsd.edu] Sent: Thursday, May 15, 2014 12:50 AM To: Grubelich, Claudette (NIH/NIGMS) [E] Cc: Nie, Zhongzhen (NIH/NIGMS) [E];Flores, Monica;Jackson, Derek;Orantes, Wilma Subject: Revised Specific Aims for Grant 2RO1 GM49711, PI: Mauricio Montal Importance: High Dear Dr. Grubelich: Attached please find the Revised Specific Aims for the above-referenced grant in pdf format. With many thanks and best wishes, Mauricio Mauricio Montal MD, PhD Distinguished Professor of Biological Sciences Section of Neurobiology University of California San Diego 9500 Gilman Drive La Jolla, CA 92093-0366 Tel: +1 858 534 0931 Fax: +1 858 822 3763

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049711-17
Application #
8625491
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Nie, Zhongzhen
Project Start
1993-08-10
Project End
2018-04-30
Budget Start
2014-06-15
Budget End
2015-04-30
Support Year
17
Fiscal Year
2014
Total Cost
$334,580
Indirect Cost
$98,580
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Syeda, Ruhma; Santos, Jose S; Montal, Mauricio (2014) Lipid bilayer modules as determinants of K+ channel gating. J Biol Chem 289:4233-43
Montal, Mauricio (2014) Redox regulation of botulinum neurotoxin toxicity: therapeutic implications. Trends Mol Med 20:602-3
Fischer, Audrey; Montal, Mauricio (2013) Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function. Toxicon 75:101-7
Santos, Jose S; Syeda, Ruhma; Montal, Mauricio (2013) Stabilization of the conductive conformation of a voltage-gated K+ (Kv) channel: the lid mechanism. J Biol Chem 288:16619-28
Fischer, Audrey; Sambashivan, Shilpa; Brunger, Axel T et al. (2012) Beltless translocation domain of botulinum neurotoxin A embodies a minimum ion-conductive channel. J Biol Chem 287:1657-61
Ogiwara, I; Ito, K; Sawaishi, Y et al. (2009) De novo mutations of voltage-gated sodium channel alphaII gene SCN2A in intractable epilepsies. Neurology 73:1046-53
Montal, Mauricio (2009) Vpu matchmakers as a therapeutic strategy for HIV infection. PLoS Pathog 5:e1000246
Santos, Jose S; Grigoriev, Sergey M; Montal, Mauricio (2008) Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel. J Gen Physiol 132:651-66
Santos, Jose S; Lundby, Alicia; Zazueta, Cecilia et al. (2006) Molecular template for a voltage sensor in a novel K+ channel. I. Identification and functional characterization of KvLm, a voltage-gated K+ channel from Listeria monocytogenes. J Gen Physiol 128:283-92
Lundby, Alicia; Santos, Jose S; Zazueta, Cecilia et al. (2006) Molecular template for a voltage sensor in a novel K+ channel. II. Conservation of a eukaryotic sensor fold in a prokaryotic K+ channel. J Gen Physiol 128:293-300

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