Calcium Channel Gamma(6) Subunits Are Unique Modulators of Low Voltage-Activated Calcium Current

The calcium channel gamma (gamma) subunit family consists of eight members whose functions include modulation of high voltage-activated (HVA) calcium currents in skeletal muscle and neurons, and regulation of AMPA receptor targeting. Cardiac myocytes express three gamma subunits, gamma 4, gamma6 and gamma7, whose function(s) in the heart are unknown. Here we compare the effects of the previously uncharacterized gamma 6 subunit with that of gamma4 and gamma7 on the low voltage-activated (LVA) calcium channels, Cav3.1 and Cav3.2, which are expressed in cardiac myocytes. We generated a bicistronic vector containing green fluorescent protein (GFP) and either the gamma6 long isofrom, the gamma6 short isoform, rat gamma4, or the recently described rat gamma7 subunits. These vectors were transiently transfected into HEK-293 cells, which had been stably transfected with either Cav3.1 or Cav3.2 alpha1 subunits. Co-expression of both the long and short isoforms of gamma6 with Cav3.1 significantly decreased LVA calcium current density in transfected cells by 49% and 69% respectively. Co-expression of gamma4 and gamma7 showed no effect. Co-expression of the long isoform of gamma6 but not gamma 4 with Cav3.2 resulted in a 54% decrease in current density in transfected cells. None of the gamma subunits studied had any effects on the voltage dependency or kinetics of Cav3.1 or Cav3.2. The gamma6, gamma 4, and gamma7 subunits are phylogenetically divergent from one another. Using a chimeric strategy we show that the functional distinction between gamma6 and gamma4 is mediated by a region within the first transmembrane domain (TM) of these proteins. The insertion of only TM1 from gamma6 into gamma4 can confer the regulatory function of gamma6 onto gamma4. Conversely, by substituting only the TM1 from gamma4 into gamma6 the calcium channel regulatory function of gamma6 can be removed. Electrophysiological analysis of a series of gamma6/gamma 4 chimeras and the use of a mammalian 2-hybrid assay confirm the functional significance of the TM1 region of gamma subunits. In addition to providing the first functional characterization of the gamma6 subunits, these results show that the effects of gamma subunits depend on both the structure of the TM1 region of the gamma subunit.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular and Integrative Physiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Hansen, Jared Paul

Contributors dc:contributor

• Best, Philip M.

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI3199205

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/87236