ISOLATION OF NEURONAL VOLTAGE SENSITIVE CALCIUM CHANNELS

THE LONG TERM OBJECTIVE OF THIS PROJECT IS TO ELUCIDATE THE STRUCTURAL AND FUNCTIONAL DIVERSITY OF NEURONAL VOLTAGE-SENSITIVE CALCIUM CHANNELS (VSCC) BY MOLECULAR CLONING AND EXPRESSION OF THE CHANNEL PROTEINS. AVAILABILITY OF PURE SUBTYPES OF NEURONAL VSCC IS CRUCIAL TO THE DEVELOPMENT OF HIGHLY SELECTIVE ANTAGONISTS WHICH HAVE GREAT POTENTIAL FOR THERAPY OF VARIOUS NEUROLOGICAL DISEASES RANGING FROM EPILEPSY TO SENILE DEMENTIA. THE STRATEGY INVOLVES THE ISOLATION OF THE CHANNEL PROTEIN WHICH BINDS CONOTOXIN BY THE USE OF ESTABLISHED PROCEDURES INVOLVING LECTIN CHROMATOGRAPHY AND AFFINITY CHROMATOGRAPHY. THE PURIFIED PROTEIN, WHICH HAS BEEN IDENTIFIED BY AFFINITY CROSS-LINKING AND PHOTO-LABELING, WILL BE ELECTROELUTED AFTER SEPARATION BY SDS GEL ELECTROPHORESIS AND SEQUENCED. OLIGONUCLEOTIDE PROBES BASED ON THE PEPTIDE SEQUENCES WILL BE USED TO SCREEN A CDNA LIBRARY OF THE ELECTRIC ORGAN OF THE RAY NARCINE BRASILIENSIS AND ISOLATE CDNA CLONES. THE LONG TERM OBJECTIVE OF THIS PROJECT IS TO ELUCIDATE THE STRUCTURAL AND FUNCTIONAL DIVERSITY OF NEURONAL VOLTAGE-SENSITIVE CALCIUM CHANNELS (VSCC) BY MOLECULAR CLONING AND EXPRESSION OF THE CHANNEL PROTEINS. AVAILABILITY OF PURE SUBTYPES OF NEURONAL VSCC IS CRUCIAL TO THE DEVELOPMENT OF HIGHLY SELECTIVE ANTAGONISTS WHICH HAVE GREAT POTENTIAL FOR THERAPY OF VARIOUS NEUROLOGICAL DISEASES RANGING FROM EPILEPSY TO SENILE DEMENTIA. THE STRATEGY INVOLVES THE ISOLATION OF THE CHANNEL PROTEIN WHICH BINDS CONOTOXIN BY THE USE OF ESTABLISHED PROCEDURES INVOLVING LECTIN CHROMATOGRAPHY AND AFFINITY CHROMATOGRAPHY. THE PURIFIED PROTEIN, WHICH HAS BEEN IDENTIFIED BY AFFINITY CROSS-LINKING AND PHOTO-LABELING, WILL BE ELECTROELUTED AFTER SEPARATION BY SDS GEL ELECTROPHORESIS AND SEQUENCED. OLIGONUCLEOTIDE PROBES BASED ON THE PEPTIDE SEQUENCES WILL BE USED TO SCREEN A CDNA LIBRARY OF THE ELECTRIC ORGAN OF THE RAY NARCINE BRASILIENSIS AND ISOLATE CDNA CLONES.