Channel proteins, a special class of membrane proteins which mediate cell signaling, are pivotal control elements of cellular homeostasis. Their dysfunction leads to disease processes and they constitute a prime target for drug intervention. The ultimate goal is to understand the fundamental principles underlying their molecular design. The immediate objective is to establish the occurrence of minimum units of structure with specific functional attributes as a tractable approach to investigate the sequence-structure determinism. The novelty of the strategy resides in the notion of a discrete modular assembly based on the premise that small, independently folded modules may be stable in the absence of the entire channel protein and that structure determination of such isolated modules reconstituted in lipid bilayers is feasible and realistic. Specifically, the program is focused on voltage- gated channel proteins. The strategy considers that given the primary structure of channel proteins it may be possible to identify functional modules associated with the ionic pore and the voltage sensor, and that such sequences may fold predictably into stable motifs that will retrieve the permeation and gating properties which are characteristic of intact channels. Sequence analysis and conformational energy calculations guide the designs. Proteins are produced by expression in bacteria of synthetic genes encoding the designed channel proteins. Channel properties are established by reconstitution of designed proteins in lipid bilayers and by heterologous expression of cDNAs encoding the proteins in amphibian oocytes. Ionic current measurements provide an assay of the permeation properties and of the voltage-dependent regulation of the probability of the channel residing in the open or closed states. Displacement current measurements probe the conformational transitions between states. Protein structure is determine by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated detergent micelles and by solid-state NMR in oriented phospholipid bilayer lamellae. Structure- function relations are developed by the convergence of structural information with the characterization of channel function. Site-specific replacements assist in refining a structure-function map. The ultimate test of a successful design is recapitulation of functional attributes of the whole protein by assembling it from independent modules. The development of a repertoire of modules, that by combination and permutation may generate functional diversity, is exciting and realistic. These discoveries may contribute clues to understand mechanisms of disease and provide structural blueprints for drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049711-06
Application #
2749942
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1993-08-10
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
6
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Ogiwara, Ikuo; Miyamoto, Hiroyuki; Tatsukawa, Tetsuya et al. (2018) Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice. Commun Biol 1:
Montal, Mauricio (2017) Tetanus neurotoxin: conformational plasticity as an adaptive strategy. EMBO Rep 18:1268-1270
Syeda, Ruhma; Santos, Jose S; Montal, Mauricio (2016) The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine. J Biol Chem 291:2931-7
Syeda, Ruhma; Qiu, Zhaozhu; Dubin, Adrienne E et al. (2016) LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength. Cell 164:499-511
Syeda, Ruhma; Xu, Jie; Dubin, Adrienne E et al. (2015) Chemical activation of the mechanotransduction channel Piezo1. Elife 4:
Montal, Mauricio (2014) Redox regulation of botulinum neurotoxin toxicity: therapeutic implications. Trends Mol Med 20:602-3
Syeda, Ruhma; Santos, Jose S; Montal, Mauricio (2014) Lipid bilayer modules as determinants of K+ channel gating. J Biol Chem 289:4233-43
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
Santos, Jose S; Asmar-Rovira, Guillermo A; Han, Gye Won et al. (2012) Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes. J Biol Chem 287:43063-70

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