Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1.
Recent evidence indicates that several members of the Na+-coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed in Xenopus oocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transport-associated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKC-mediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.
Quick
M W
MW
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021, USA.
Corey
J L
JL
Davidson
N
N
Lester
H A
HA
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D026901:Membrane Transport Proteins; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters; D011506:Proteins / Q000502:physiology*; D051381:Rats; D015290:Second Messenger Systems / Q000502:physiology*
D000596:Amino Acids; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D011506:Proteins; C493415:Slc6a1 protein, rat