REGULATION OF L-TYPE VOLTAGE-DEPENDNET CALCIUM CHANNELS BY THE REM GTPASE

Author

Chunyan Pang, University of KentuckyFollow

Date Available

12-14-2011

Year of Publication

2008

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. Douglas A. Andres

Second Advisor

Dr. Peter. H. Spielmann

Abstract

The Rem, Rem2, Rad, and Gem/Kir GTPases, comprise a novel subfamily of the small Ras-related GTP-binding proteins known as the RGK GTPases, and have been shown to function as potent negative regulators of high voltage-activated (HVA) Ca²⁺ channels upon overexpression. HVA Ca²⁺ channels modulate Ca²⁺ influx in response to membrane depolarization to regulate a wide variety of cellular functions and they minimally consist of a pore-forming α1 subunit, an intracellular β subunit, and a transmembrane complex α₂/δ subunit. While the mechanisms underlying RGK-mediated Ca²⁺ channel regulation remain poorly defined, it appears that both membrane localization and the binding of accessory Ca²⁺ channel β subunits (Ca_Vβ) are required for suppression of Ca²⁺ channel currents. We identified a direct interaction between Rem and the L-type Ca_vα₁ C-terminus (CCT), but not the CCT from Ca_V3.2 T-type channels. Deletion mapping studies suggest that the conserved CB-IQ domain is required for Rem:CCT association, a region known to contribute to both Ca²⁺-dependent channel inactivation and facilitation through interactions of Ca²⁺-bound calmodulin (CaM) with the proximal CCT. Furthermore, both Rem2 and Rad GTPases display similar patterns of CCT binding, suggesting that CCT represents a common binding partner for all RGK proteins. While previous studies have found that association of the Rem C-terminus with the plasma membrane is required for channel inhibition, it is not required for Ca_Vβ- subunit binding. However, Rem:CCT association is well correlated with the plasma membrane localization of Rem and more importantly, Rem-mediated channel inhibition upon overexpression. Moreover, co-expression of the proximal CB-IQ containing region of CCT (residues 1507-1669) in HIT-T15 cells partially relieves Rem blockade of ionic current. Interestingly, Ca²⁺/CaM disrupts Rem:CCT association in vitro. Moreover, CaM overexpression partially relieves Rem-mediated L-type Ca²⁺ channel inhibition and Rem overexpression alters the kinetics of calcium-dependent inactivation. Together, these data suggest that the association of Rem with the CCT represents a crucial molecular determinant for Rem-mediated L-type Ca²⁺ channel regulation and provides new insights into this novel channel regulatory process. These studies also suggest that instead of acting as complete Ca²⁺ channel blockers, RGK proteins may function as endogenous regulators for the channel inactivation machinery.

Recommended Citation

Pang, Chunyan, "REGULATION OF L-TYPE VOLTAGE-DEPENDNET CALCIUM CHANNELS BY THE REM GTPASE" (2008). University of Kentucky Doctoral Dissertations. 656.
https://uknowledge.uky.edu/gradschool_diss/656