High-voltage-activated calcium (CaV1/CaV2) channels are necessary for the function of excitable cells. Molecules that inhibit CaV1/CaV2 channels powerfully regulate physiology, and are important or potential therapeutics for many serious diseases including: hypertension, neuropathic pain, cardiac arrhythmias, and Parkinson's disease. CaV1/CaV2 channels are potently inhibited by a four-member family of monomeric G- proteins known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. RGKs are expressed in excitable tissues, and their expression level often changes correlatively with disease, suggesting their strong regulation of CaV1/CaV2 has broad patho-physiological implications. Engineered RGKs have potential therapeutic applications as genetically-encoded CaV channel blockers (CCBs) for a broad range of diseases. For specific applications, genetically encoded inhibitors may provide a higher therapeutic index than traditional small molecule CCBs because they can be locally expressed, thereby achieving effective CaV channel block while minimizing off- target effects. The precise molecular mechanisms by which RGKs inhibit CaV1/CaV2 channels are not well- understood. Our preliminary data hint at a surprising degree of customization and complexity where distinct RGK proteins differentially use multiple mechanisms and structural determinants to inhibit individual CaV1/CaV2 channel isoforms. Precise understanding of the mechanisms underlying customized RGK inhibition of CaV1/CaV2 channels is critical for insights into the patho-physiological ramifications of this channel regulation, as well as efforts to engineer useful new genetically-encoded CCBs. Our long-term objective is to furnish fundamental understanding of the diverse molecular mechanisms and structural determinants underlying RGK inhibition of CaV1/CaV2 channels and to bridge these insights to: (i) a new appreciation of the patho-physiological implications of this channel modulation; and (ii) the design of novel, useful genetically-encoded CCBs as potential therapeutics. We combine whole-cell and single-channel electrophysiology, fluorescence resonance energy transfer (FRET), molecular biology, channel engineering, and biochemical approaches to address three specific Aims: (1) Dissect mechanisms the RGK protein, Rem, uses to inhibit recombinant CaV1.2 channels. (2) Determine and contrast mechanisms of RGK inhibition across the CaV1/CaV2 channel family. (3) Dissect mechanisms of RGK inhibition of native CaV1.2 channels in cardiomyocytes.

Public Health Relevance

The proposal focuses on understanding mechanisms of small G-protein inhibition of voltage-dependent calcium channels and using them as inspiration to develop novel genetically encoded calcium channel inhibitors. Such bio-inspired molecules have potential therapeutic applications for treating neurological diseases including Alzheimer's disease, pain, and Parkinson's disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM107585-04
Application #
9247954
Study Section
Special Emphasis Panel (ZRG1-MDCN-N (04)M)
Program Officer
Nie, Zhongzhen
Project Start
2014-04-01
Project End
2018-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
4
Fiscal Year
2017
Total Cost
$273,600
Indirect Cost
$102,600
Name
Columbia University (N.Y.)
Department
Physiology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
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