Charge compensation mechanism of a Na+-coupled, secondary active glutamate transporter.

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.

Mechanism of cation binding to the glutamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101.

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na(+) ions, one H(+) ion, and one glutamate molecule into the cell, in exchange for one K(+) ion. Na(+) binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na(+)] dependence of this conductance as a read-out of Na(+) binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr(101), in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na(+) was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1(WT), in the T101A mutant this [Na(+)] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na(+) ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na(+) binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr(101) and Asp(367). A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.

Sequence selective recognition of double-stranded RNA at physiologically relevant conditions using PNA-peptide conjugates.

Conjugation of short peptide nucleic acids (PNA) with tetralysine peptides strongly enhanced triple helical binding to RNA at physiologically relevant conditions. The PNA hexamers and heptamers carrying cationic nucleobase and tetralysine modifications displayed high binding affinity for complementary double-stranded RNA without compromising sequence selectivity. The PNA-peptide conjugates had unique preference for binding double-stranded RNA, while having little, if any, affinity for double-stranded DNA. The cationic PNAs were efficiently taken up by HEK293 cells, whereas little uptake was observed for unmodified PNA.

Protonation state of a conserved acidic amino acid involved in Na(+) binding to the glutamate transporter EAAC1.

Substrate transport by glutamate transporters is coupled to the co-transport of 3 Na(+) ions and counter-transport of 1 K(+) ion. The highly conserved Asp454, which may be negatively charged, is of interest as its side chain may coordinate cations and/or contribute to charge compensation. Mutation to the nonionizable Asn resulted in a transporter that no longer catalyzed forward transport. However, Na(+)/glutamate exchange was still functional, as demonstrated by the presence of transient currents following rapid substrate application and voltage jumps. While the kinetics of Na(+)/glutamate exchange were slowed, the apparent valence (z) of the charge moved in EAAC1 D454N (0.71) was similar to that of EAAC1 WT (0.64). Valences calculated using the Poisson-Boltzmann equation were close to the experimental values for EAAC1 D454N (0.55), and with D454 protonated (0.45). In addition, pK(a) calculations performed for the bacterial homologue GltPh revealed a highly perturbed pK(a) (7.6 to >14) for D405 residue (analogous to D454), consistent with this site being protonated at physiological pH. In contrast to the D454N mutation, substitution to alanine resulted in a transporter that still bound glutamate, but could not translocate it. The results are consistent with molecular dynamics simulations, showing that the alanine but not the asparagine mutation resulted in defective Na(+) coordination. Our results raise the possibility that the protonated state of D454 supports transporter function.