Mutating a conserved proline residue within the trimerization domain modifies Na+ binding to excitatory amino acid transporters and associated conformational changes.
Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.
From the Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover Germany.
Jasmin
J
Hotzy
Nicole
N
Schneider
Peter
P
Kovermann
Christoph
C
Fahlke
D000818:Animals; D017124:Conserved Sequence; D027341:Excitatory Amino Acid Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D011392:Proline / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism*; D014981:Xenopus
D027341:Excitatory Amino Acid Transporter 1; C493394:SLC1A3 protein, human; D018698:Glutamic Acid; D011392:Proline; D012964:Sodium
Anion Transport; Ataxia; Glutamate; Neurological Diseases; Neurotransmitter Transport; Voltage Clamp Fluorometry