An Oocyte Expression/Patch Clamp Analysis System for the Study of Membrane Channel Proteins

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Several research groups at Rutgers/UMDNJ have focused on the molecular characterization of newly-identified membrane proteins (channels, receptors, transporters) in animal and plant systems. Use of molecular genetic approaches has led to the cloning of putative cDNAs encoding polypeptide components of these membrane proteins. Rapid advancement of the research programs characterizing these cDNAs will, in the near future, depend on the capability to characterize the transport functions facilitated by the translation products of the cDNAs. One state-of-the-art technology which can be employed to facilitate this characterization is oocyte expression/patch clamp analysis. This involves the microinjection of cRNA transcribed from these cDNAs into frog (Xenopus laevis) oocytes, leading to translation and functional expression of the transport proteins in oocyte membranes. Monitoring of ion currents across the plasmalemma of oocytes expressing these proteins by use of patch/voltage clamp equipment can then be employed to study the specific transport functions of these gene products. This proposal seeks funds to set up the first oocyte expression/patch clamp analysis system at Rutgers. The research programs described in this proposal will all make use of this equipment to gain insights into the structure/function relationships of the target membrane proteins. The specific research objectives which will be perused by investigators using the proposed equipment are as follows. 1 ) lon channel genes heretofore not known to be present in plants will be characterized. Specifically, cDNAs encoding a subunit of plant K+ channels, a ligandgated type 11 (30-40 kD polypeptide) K+ channel native to an intracellular plant membrane, and a K+ channel which has a unique pore sequence (unknown previously in plant or animal membranes) and is a likely candidate for the Na+ uptake pathway in plants will be studied. 2) Polypeptide components of the enzyme complex involved in plant ce ll wall callose (cellulose) biosynthesis will be characterized. The cDNAs encoding these polypeptides share sequence homology with members of the MIP (major intrinsic protein) or aquaporin gene family. 3) A cDNA encoding a novel member of the family of glutamate (Glu) family of excitatory amino acid receptors will be characterized. Glu receptors (GluRs) mediate rapid excitatory neurotransmission in the mammalian central nervous system. GluRs are a type of cation channel known as ionotropic receptors. The cloned cDNA encodes a unique splice variant that is a truncated receptor form of a GluR. 4. Nuclear-encoded proteins are imported into plant cell chloroplasts via specific channels spanning the double membrane delimiting the organelle. A complex of six chloroplast envelope membrane proteins have been identified as components of the chloroplast protein import machinery. A cDNA encoding one of these proteins, IAP75, has been cloned. Sequence analysis of IAP75 indicates that it has secondary structure similarity to channel proteins such as bacterial outer membrane porins. The porinlike nature of IAP75 will be characterized.