Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.

Ion flow in many voltage-gated K؉ channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K ؉ channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe 551 to Trp 563 , affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala 561 , His 562 , Ala 565 , Trp 568 , and Ile 571 ), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.

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 gly-cine 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 [ 3 H]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.

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The hERG K(+) channel undergoes rapid inactivation that is mediated by 'collapse' of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600-642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two-four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K(+) channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.