The overall interest of my laboratory is the regulation of synapse formation. The specific system that we use to address questions in this area is the mammalian neuromuscular junction. This synapse has proved to be a model experimental approach for elucidating the biochemical mechanisms that underlie such processes as ion channel regulation and signal transduction at synapses. In particular, we have focused our efforts on studying the regulation of phosphorylation of the nicotinic acetylcholine receptor (nAChR). The (nAChR) is a ligandgaited ion channel that mediates signal transduction at the neuromuscular junction. We have recently demonstrated that the nAChR autoregulates its own state of phosphorylation by conducting calcium ions into the cell in response to agonist activation of the ion channel. We believe this change in nAChR phosphorylation may potentially alter ion channel properties of the receptor and thereby modulate synaptic transmission at this synapse.
We have since become interested in characterizing calcium sensitive protein kinases that may play a role in the neuromuscular junction. Our attention has been directed at the protein kinase C (PKC) family. PKC is a family of protein serine/threonine kinases consisting of multiple isoforms. In an effort to distinguish the physiological roles of two isoforms, PKC that are highly enrich in rat skeletal muscle, we examined their subcellular distribution, developmental expression and intracellular localization.
Our results suggest that nPKC q, rather than the nPKC a, is involved in physiological functions that specific for skeletal muscle. Immunoreactivity for nPKC q was highest in the membrane subcellular fraction compared to the cytosolic fraction of skeletal muscle. In contrast, cPKC was found to be predominantly distributed in the cytosolic rather than membrane fraction. PKC q appeared to be developmentally regulated postnatally in rat skeletal muscle with a 4fold increase in expression occurring exclusively in the membrane fraction during postnatal days 3 through 21. This time course coincides with the period in rat development associated with maturation of neuromuscular junctions.
The intracellular localization of the PKC q and a isoforms in rat skeletal muscle was examined by immunocytochemistry. PKC q was detected in association with sarcolemma of skeletal muscle and was found to be localized in the neuromuscular junction. taken together, these data suggest that nPKC q may play a specific role in skeletal muscle signal transduction in both the developing and mature neuromuscular synapse.
RECENT PUBLICATIONS
Miles, K., S. M. Audigier, P. Greengard and R. L. Huganir, Autoregulation of phosphorylation of the nicotinic acetylcholine receptor. The Journal of Neuroscience 114:32713279, 1994.
Cala, S. F. and K. Miles, Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle. Biochimica et Biophysica Ada. 1118:277287, 1992.
KATHYRN MILES, Ph.D.
Assistant Professor
Department of Anatomy and Cell Biology
Phone: 270-3889/1443, Fax: 270-3732
E-mail: kmiles@downstate.edu
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