Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

Voltage-gated calcium channels mediate the influx of Ca 2؉ ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming ␣ 1 -subunit and the auxiliary ␤-and ␣ 2 ␦-subunits. The ␤-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four ␤-subunit genes have been cloned, ␤ 1-4 , with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The ␤ 1b -subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming ␣ 1 -and the regulatory ␤-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the ␤ 1b -subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The ␤ 1b -subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the ␣ 1 -subunit (the ␣-interaction domain). Using the cutopen oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac ␣ 1 -subunit (␣ 1C ) co-injected with folded-␤ 1b -protein or ␤ 1b -cRNA. We demonstrate that the co-expression of the ␣ 1C -subunit with either folded-␤ 1b -protein or ␤ 1b -cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the ␤ 1b -subunit primarily modulates channel activity, rather than expression.Changes in the intracellular calcium concentration regulate a variety of cellular functions such as neurotransmission, muscle contraction, hormone secretion, and gene expression. High threshold voltage-activated calcium channels are the main route for calcium entry in electrically excitable cells. They are membrane protein complexes composed of at least three nonhomologous subunits, the ␣ 1 -, ␤-, and the ␣ 2 /␦-subunit. Through molecular cloning, at least 10 genes encoding mammalian ␣ 1 -subunits (␣ 1A-I and ␣ 1S ) have been identified in different cell types (1). Although the ␣ 1 -subunit encompasses all the structural elements of a functional voltage-activated calcium channel, such as the ion-conduction pathway, the voltage sensor, and drug-binding sites, the ␤-subunit seems to be essential for channel performance (2) and to be acting at two levels: (i) channel expression by interfering with the ␣ 1 -subunit endoplasmic reticulum (ER) 1 -retention signal to facilitate intracellular trafficking (3, 4), and (ii) channel activity by modifying the electrophysiological properties of the channel (5-7).Two highly conserved sequences have been identified as the primary interaction site between the ␣ 1 -and the ␤-subunit, the ␣ 1 -subunit interaction domain (AID) that lies within the c...