Previously Postdoctoral Research Associate, Pharmacology and Toxicology
University of Kansas
Trafficking of the Plasma Membrane CA2+-ATPase to Rafts
Rafts are cholesterol/sphingolipid rich compartments in the plasma membrane that serve as mobile platforms floating in a sea of glycerolipids, that constitute the rest of the membrane. Neuronal rafts serve as local sites for the orchestration of a variety of cellular signaling processes. Compelling evidence has emerged in the last few years implicating rafts in synaptic transmission and Ca2+ signaling but there is no information on how rafts accomplish this function and which Ca2+ regulatory proteins mediate the Ca2+ signal in rafts.
Since rafts have been implicated to be intimately involved in local Ca2+ signaling we hypothesize that these microdomains are enriched in the high affinity Ca2+ transporting enzyme, the plasma membrane Ca2+-ATPase (PMCA), particularly the faster responding isoforms possessing membrane-anchoring motifs such as PDZ domains that may serve as a potential targeting mechanism. The first step towards this project was to isolate authentic raft domains separated from the non-raft portions of the PM. We purified rafts from neuronal synaptic plasma membranes by Triton X-100 extraction under cold conditions followed by sucrose density gradient centrifugation. Rafts appeared as a distinct band of white material at the interface of fractions 3 and 4. The purity of the preparations was monitored by probing density gradient fractions with antibodies to the raft marker protein flotillin. Immunoblotting of the rafts indicated that both PMCA and its activator protein calmodulin are enriched in rafts further suggesting their involvement in local Ca2+ signaling processes orchestrated in these microdomains.
Currently, we are in the process of determining the specific isoforms and splice variants of PMCA localized within rafts and whether PDZ domain containing PMCAs are specifically found in these domains. Future goals are to identify the protein partners associated with PMCA and to determine the mechanisms involved in the trafficking of PMCA into and out of rafts.
Dr. Zaidi accepted a position as associate professor of Biochemistry at the Kansas City University of Medicine and Biosciences in 2007.