I got a grant!

I got a grant for my ion channel research project! Receiving the graduate research grant award has immensely boosted my morale, and has encouraged me towards the scientific career path that I wish to pursue.

On behalf of the Graduate Studies Office, I am pleased to inform you that you are a recipient of a Graduate Research Grant for the 2014-2015 Academic Year. Those who evaluated your proposal were impressed with the project.  We congratulate you and look forward to your successful completion of the proposed research. We will be interested in your progress as you move along in your research, and look forward to hearing your presentation at Student Research Day next spring.

The Graduate School seeks, through such awards, to encourage the highest standards of scholarship among its graduate students and we are extremely gratified to provide this opportunity for you to pursue your theory.

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Our lab finally got a new fluorescence/chemiluminescence imager and it has been great for our investigations so far. I have also been getting many positive western blot results. Evaluating the right antibody concentrations for the blots and tweaking the immunoprecipitation protocols to fit my experiments was a considerable amount of work. My next goal is to quantify the protein bands and figure out the statistical significance of GIRK channel subunit interactions. I am so glad to have had worked on it during the fall semester. I can now concentrate on putting my findings together by the time I graduate in spring(!).

Besides this, I am looking forward to working on the electrophysiology rig with my lab mates next semester. We have been concentrating on the biochemical experiments since the past couple of months. It would be interesting to perform preliminary patch clamping experiments on the cells expressing the mutated and wildtype channel subunits.

I finally got down to updating this space after a long break. I have been very busy with the research work, classes and teaching. It feels great to finally break the writing dry-spell.

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G protein-coupled inwardly-rectifying potassium (GIRK) channels

My current Master’s thesis research is focussed on understanding the structural and functional properties of G protein-coupled inwardly-rectifying potassium (GIRK or Kir 3.x) channels. This class of potassium ion channels are responsible for regulating the heart rate and modulating the neuronal excitability of certain neurons.

GIRK channels are activated by G-protein coupled receptors (GPCRs) including the muscarinic, dopamine, serotonin, GABA, opiod, and acetylcholine receptors, which are involved in many signal transduction pathways in the cell. The activation of a GPCR by its ligand (neurotransmitter or hormone) results in the release of Gα and Gβγ, two intracellular effector molecules. The activated Gβγ binds to the GIRK channel and opens it up to potassium ions resulting in the hyperpolarization of the cell (increased negative charge due to efflux of K+ ions).

Activation of GIRK channel
GIRK channels are activated upon GPCR stimulation by direct interaction with Gβγ.

Molecular cloning techniques have led to the discovery of four channel subunits – GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), GIRK3 (Kir 3.3) and GIRK 4 (Kir 3.4). GIRK1 through 3 can be found in the central nervous system and GIRK4 is primarily found in the heart. Four of these subunits assemble either as homomers or heteromers (in 1:1 subunit ratio) to form a tetrameric functional channel.

Structurally, the channel is divided into cytoplasmic and transmembrane domains. The amino- (NH2) terminus and the carboxyl- (COOH) terminus are present in the cytoplasm and contribute to the formation of the intracellular/cytoplasmic domain. Each subunit is composed of two transmembrane domains separated by a P-loop containing the “ion-selectivity filter”. This type of channel assembly results in significant interactions between the cytoplasmic domains of the four subunits.

Crystal Structure of GIRK channel. Left - Front view of four GIRK2 subunits (color coded) channel assembly. Right - Top view of the cytoplasmic domain forming the selectivity filter.
Crystal Structure of GIRK channel. Left – Front view of the channel comprised of 4 subunits (color coded). Right – Top view of the cytoplasmic domain forming the selectivity filter. Protein Data Base ID – 3SYQ and 2QKS (Whorton, M. R., Mckinnon, R., 2011)

I have been specifically involved in understanding how certain amino acid residues residing in the hydrophobic pockets of the subunits influence channel activation and function. I use multiple experimental methods to investigate the interaction between the N- and C- termini of the GIRK1 and GIRK4 channel subunits to analyse protein expression and domain association. Previous research (Sarac et al, 2005) has revealed that certain mutations in the amino acid residues of these two subunits alters channel function.

Understanding how the interaction between the different GIRK channel subunits influences the channel formation and activity is critical for the elucidation of certain cellular mechanisms involved in cell physiology as well as in various channelopathies. New studies also suggest that ethanol binds to a hydrophobic pocket in the channel and activates it. (Bodhinathan, K., Slesinger, P. A., 2013 and Aryal, P., et al, 2009) Ethanol activation of the channel can be utilised for developing selective therapeutics to treat alcohol-related disorders like alcohol addiction and abuse.

Selected Resources: