The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. This is precisely the gap in knowledge the program intends to fill. The ultimate goal of this endeavor is the understanding of the mechanism of voltage sensing based on the modular design of voltage-gated channel proteins. Major objectives are: to define the protein fold(s) best suited to fulfill the pivotal function of voltage sensing;to delineate a minimum set of determinants sufficient for sensing;to uncover a molecular blueprint for a versatile voltage sensor design for which a finite number of specified perturbations would adapt it to sense a wide range of membrane potential;and to establish the surface compatibility underlying the interaction between the two modules and the propagation of change from one module to the other that produces the exquisite sensitivity of the pore to voltage in intact voltage-gated channels. We propose to characterize the channel properties of the isolated voltage sensor module (VSM), the pore module (PM), and the self-assembled [VSM-PM] complex by overexpression and reconstitution into lipid bilayers and giant proteoliposomes, aiming to recapitulate the functional features of the intact voltage-gated K+ channel (Kv) from its component modules. We propose to explore the voltage sensor sequence landscape approached by generating and screening random libraries of VSM mutants aiming to identify and demonstrate unsuspected channels with new voltage-gating phenotypes. We intend to determine the atomic resolution-structures of KvLm and its modules by X-ray crystallography. Exciting results have already emerged which pave the way for a decidedly productive phase of the program. Overall, the itinerary entails going from modules to sequence, to structure and back to mechanism. This focused and realistic program outlines a novel way of thinking about voltage sensing.

Public Health Relevance

Ion channels, a special class of membrane proteins that allow the selective and regulated diffusion of ions across membranes, are fundamental for cell function and regulation. Their design is a marvel of protein chemistry and evolution, and their dysfunction is at the root of devastating human diseases such as epilepsy and arrhythmia. The voltage sensor, the unique element that endows Na+ and K+ channels, which underlie the nerve action potential with the exquisite sensitivity to transmembrane voltage, remains enigmatic and needs further study. The thrust of our program aims to establish structure-function relationships with a primary emphasis on the modular design of the transmembrane domain in voltage-gated channel proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049711-15
Application #
8204414
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Deatherage, James F
Project Start
1993-08-10
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
15
Fiscal Year
2012
Total Cost
$317,574
Indirect Cost
$112,025
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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