Novel mechanisms of v-ATPase regulation in vertebrates

Vacuolar H+-ATPases (v-ATPases) are ATP-driven proton pumps that acidify intracellular compartments. Acidification is important for a plethora of cellular processes, ranging from neurotransmitter uptake to lysosomal degradation. The timing of acidification is essential for pathways where lowering of the luminal pH is vital, e.g. virus/protein activation, cargo-receptor separation. Despite decades of research, key open questions persist: How is v-ATPase regulated during a process that replenishes synaptic vesicles (SVs) and sustains neurotransmission? Is lysosomal pH dynamically regulated - if yes, how? What are the consequences of poorly regulated acidification for cellular homeostasis and health? We will tackle these questions through Rabconnectin-3a, a conserved brain-enriched protein whose mutations cause mental retardation, neuropathy and/or hearing loss. Little is known about Rabconnectin-3a due to its large size, lack of tools and early embryonic lethality. We cloned Dmxl2 gene encoding Rabconnectin-3a, generated mouse models and a specific antibody, and detected Rabconnectin-3a on all organelles that acidify. I propose to examine a role of Rabconnectin-3a in the regulation of acidification, and to characterize its functions at the synapse and in two disorders. A multi-disciplinary approach will be employed, from studies of individual organelles to a characterization of neuronal networks by a combination of genetics and electrophysiology.