Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

Abstract

Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

A K+/Na+ co-binding state: Simultaneous versus competitive binding of K+ and Na+ to glutamate transporters

“…Although all three subtypes showed evidence for the anion-conducting K ϩ /Na ϩ co-binding state, K ϩ activated anion current in EAAT-1 and -2 in the absence of Na ϩ but not in EAAC1 (rat homolog of EAAT3). These results suggest subtle differences in the way the anion conductance operates between the subtypes, consistent with results on differences in Na ϩ dependence of the anion conductance (24). These differences may be interesting to explore and may be linked to the different microenvironments of these transporters in their particular location of expression, which varies among the subtypes (22)(23)(24).…”

“…However, this simplified model was later revised after it was shown for EAAT4 (30) that the anion conductance can be activated in several other states of the transport cycle, including the inward-facing, K ϩ -bound state that is responsible for K ϩ -induced relocation of the transporter. In addition, Amara and co-workers (24) showed that the glial glutamate transporter subtypes EAAT1 and -2 do not require Na ϩ to acti- Glutamate transporter K ؉ /Na ؉ co-binding state vate the leak anion conductance in contrast to the neuronal subtypes EAAT3 and -4. This finding further supported the idea that the anion conductance is present in many states of the transport cycle, including Na ϩ -free states, and is modulated as the transporter moves through the transport cycle.…”

“…This anion current component was termed the leak anion current. Although the leak anion current requires the presence of sodium ions in the solution for the neuronal subtypes EAAT3 (8) and -4 (22), it is sodium-independent for the glial subtypes EAAT1 (23) and -2 (24). This finding suggests that subtle differences exist among the members of the SLC1 family with respect to the mechanism of anion conductance activation.…”

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates

“…2, C and D). EAAT2 also mediates large anion current amplitudes under conditions, in which extracellular Na + is completely substituted by K + (36,37). WT and mutant currents displayed similar time, voltage and substrate dependences.…”

Functional Astrocyte Heterogeneity and Implications for Their Role in Shaping Neurotransmission

“…In addition, although glutamate transport results in the import of a net positive charge in both cases, the reversal potential of the current generated by GLAST activity is more negative than GLT-1 and is altered by extracellular chloride concentrations (Wadiche and Kavanaugh, 1998 ). This difference in ion flux is likely due to differences in a secondary property of glutamate transporters—their ability to conduct anions, independent of their transport activity (Wadiche and Kavanaugh, 1998 ; Machtens et al, 2015 ; Divito et al, 2017 ). This property of glutamate transporters is often overlooked by studies examining transporter function in astrocytes, but there is evidence that chloride efflux through glutamate transporter-formed channels greatly influences intracellular chloride concentrations in cerebellar Bergmann glia (Untiet et al, 2017 ).…”