Cholesterol is required for the reconstruction of the sodium- and chloride-coupled, gamma-aminobutyric acid transporter from rat brain.
The reconstruction of the purified sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain into asolectin liposomes requires the addition of brain lipids (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865). The reconstitution assay was used to identify the component(s) from brain lipids responsible for the stimulation during the fractionation of brain lipids. The distribution of the active component was found to be similar to that of cholesterol. Furthermore, cholesterol was found to mimic the effect of brain lipids and it stimulated the transport activity up to 20-fold. Optimal reconstituted transport activity was achieved with mixtures of cholesterol and any one of several phospholipids, such as phosphatidylcholine, phosphatidylserine or phosphatidylglycerol. gamma-Aminobutyric acid transport in these liposomes of defined composition exhibited all the properties of the native transporter, such as the absolute dependence on sodium and chloride and electrogenicity. Cholesterol could not be replaced by cholest-4-en-3one and other steroids, and thus its effect is probably not due to effects on membrane fluidity. The requirement was also not due to effects on intactness of the liposomes or incorporation of proteins into them. Furthermore it was found that the reconstitution of the sodium and potassium coupled L-glutamic acid transporter from rat brain also required cholesterol. However, in this case the optimal activity was reached by 4-5-fold lower levels of cholesterol than those necessary for gamma-aminobutyric acid transport. When cholesterol depletion from the transporters was incomplete, addition of exogenous brain lipids was not required. Thus, if the cholesterol was still associated with the transporter proteins, its final concentration, as a fraction of the total lipids present in the reconstitution mixture, was only about 0.01 mol%. Thus, it is likely that the effects of cholesterol are due to direct interactions with the cotransporters and not to an average effect on membrane properties.
Shouffani
A
A
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Kanner
B I
BI
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D002784:Cholesterol / Q000031:analogs & derivatives / Q000494:pharmacology*; D050485:GABA Plasma Membrane Transport Proteins; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids / Q000302:isolation & purification; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D027361:Organic Anion Transporters; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D013329:Structure-Activity Relationship; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D005680:gamma-Aminobutyric Acid; C024990:asolectin; D002784:Cholesterol; D012964:Sodium