Date of Degree
PhD (Doctor of Philosophy)
Robert A. Cornell
The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.
dopaminergic, melanophore, Parkinson's, TRPM7, Zebrafish
Copyright 2009 Matthew S. McNeill