Molecular basis for proton regulation of glycine transport by glycine transporter subtype 1b

In the central nervous system, glycine is a coagonist with glutamate at the N-methyl-D-aspartate subtype of ionotropic glutamate receptors. The GLYT1b subtype of glycine transporters is expressed in similar regions of the brain as the excitatory N-methyl-D-aspartate receptors and has been postulated to regulate glycine concentrations within excitatory synapses. We have expressed GLYT1b in Xenopus laevis oocytes and used electrophysiological techniques to investigate the pH regulation of glycine transporter function. We found that H+ inhibits glycine transport by a noncompetitive mechanism, with half-maximal inhibition occurring at concentrations found in both physiological and pathological conditions. Charge-to-flux experiments revealed that the decreased current measured corresponds to a decreased influx of [H-3] glycine and that the proton inhibition of GLYT1b does not alter the coupling ratio of transport. The membrane potential does not affect proton inhibition of transport, suggesting that the site of action on GLYT1b is not within the electric field of the membrane. Mutation of histidine 421 to an alanine residue, in the fourth extracellular loop of GLYT1b, renders the transporter insensitive to regulation by pH, but does not seem to alter the kinetics of glycine transport. These results suggests that histidine 421 is responsible for mediating the inhibitory actions of protons. Proton modulation of GLYT1b may be an important factor in determining the dynamics of excitatory neurotransmission.