The substrates of a sodium- and chloride-coupled gamma-aminobutyric acid transporter protect multiple sites throughout the protein against proteolytic cleavage.
Fragments of the (Na(+) + Cl-)-coupled GABAA transporter were produced by proteolysis of membrane vesicles and reconstituted preparations from rat brain. The former were digested with Pronase, the latter with trypsin. Fragments with different apparent molecular masses were recognized by sequence-directed antibodies raised against this transporter. When GABA was present in the digestion medium, the generation of these fragments was almost entirely blocked. At the same time, the neurotransmitter largely prevented the loss of activity caused by the protease. The effect was specific for GABA; protection was not afforded by other neurotransmitters. It was only observed when the two cosubstrates, sodium and chloride, were present on the same side of the membrane as GABA. The results indicate that the transporter may exist in two conformations. In the absence of one or more of the substrates, multiple sites located throughout the transporter are accessible to the proteases. In the presence of all three substrates--conditions favoring the formation of the translocation complex--the conformation is changed such that these sites become inaccessible to protease action.
Mabjeesh
N J
NJ
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Kanner
B I
BI
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050485:GABA Plasma Membrane Transport Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027361:Organic Anion Transporters; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011402:Pronase / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D010446:Peptide Fragments; D010455:Peptides; D011510:Proteolipids; C020485:proteoliposomes; D005680:gamma-Aminobutyric Acid; D012964:Sodium; D011402:Pronase