The Calcium Channel Gamma6 Subunit Analysis of Function and Determination of a Sequence Motif Critical for Its Effect

The auxiliary gamma6 subunit of voltage dependent calcium channels is known to decrease calcium current density when co-expressed with the pore forming Cav3.1 subunit. In this study, I showed that Cav3.1 calcium channels are a major pathway for calcium influx at resting membrane potential, and that the gamma6 subunit is a modulator of this LVA window current in HEK-Cav3.1 cells by employing a calcium imaging technique. Further, I demonstrated that co-expression of gamma6 subunit decreases Cav2.3 dependent calcium current density. Previous studies using chimeric gamma subunits indicate that the N-terminal region, including the first transmembrane domain (TM1), is critical for the inhibitory function of the gamma6 subunit. In this study, I have investigated the functional properties of the gamma6 TM1 and identified critical motifs and residues in this region. Calcium current density in HEK-Cav3.1 cells was monitored following transfection with plasmids containing either gamma 6 TM1 or various mutants of this peptide. The gamma6 TM1 significantly inhibits the expression of Cav3.1 current suggesting that gamma 6 TM1 is both necessary and sufficient to inhibit Cav3.1 calcium current. Co-immunoprecipitation experiments indicate the presence of a gamma 6/alpha1 subunit complex when the two proteins are co-expressed. We identified two adjacent GxxxA motifs (G42xxxA46xxG 49xxxA53) in the gamma6 TM1 which are predicted to produce a long groove on one face of the helix due to the short side chains of the glycine and alanine residues. GxxxA and related motifs are thought to be important for promoting and stabilizing helix-helix interactions. Therefore we performed site directed mutagenesis of these specific residues in TM1 replacing the G and A residues with amino acids containing large side chains. The G42L and A46I mutants are no longer inhibitory while the G49L mutant retained the inhibitory function of the wild type. Our results suggest that the first GxxxA motif within TM1 of gamma6 is critical for its ability to inhibit Cav3.1 calcium current. Overall, my study enhances our understanding of the function and structure of the gamma6 subunit, and provides a basic approach to design novel therapeutic agents to target calcium channels.

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

• Lin, Zuojun

Contributors dc:contributor

• Philip Best

• Biophysics, General

Language dc:language

eng

Identifier

(MiAaPQ)AAI3202130

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/87239