Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Anatomy and Cell Biology

Supervisor

Dr. John F. MacDonald

2nd Supervisor

Dr. Michael F. Jackson

Joint Supervisor

Abstract

TRPM2 (1507 amino acids), a non-selective cation channel with substantial permeability for Ca2+, is responsive to oxidative stress, and is a mediator of cell death in several cell types. Ca2+-calmodulin has been shown to promote channel activation and inactivation, however the mechanisms are not fully understood. Identifying candidate CaM binding sites using in silico screening, I hypothesized that Ca2+-dependent inactivation (CDI) of TRPM2 is mediated by an intracellular CaM binding domain unique from that of activation (406-415AA). I systematically determined the minimum binding domains for three CaM candidate sites on TRPM2’s intracellular domains using truncated fragments and subsequent CaM-Sepharose pull-downs. TRPM2 with substitution mutations to candidate sites were transfected into HEK293 cells; currents were recorded using 2mM or 0.5mM Ca2+ extracellular fluid and adenosine diphosphate ribose (ADPR) in the patch pipette. Abolished and reduced currents respectively were observed as a result of amino acid substitution to CaM binding regions at 172-187AA and 1087-1101AA of TRPM2. The two identified CaM candidate sites may establish a potential molecular link to CDI of TRPM2.

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