Master of Science
Anatomy and Cell Biology
Dr. John F. MacDonald, Dr. Michael F. Jackson
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.
Lockhart, Brian M. W., "Calclium-calmodulin regulation of TRPM2 currents" (2012). Electronic Thesis and Dissertation Repository. 663.