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.

NCLX/NCKX6 is a recently identified gene responsible for a mitochondrial Na þ-Ca 2þ exchanger (NCX mit), a Ca 2þ extrusion system from mitochondria. Although there have been accumulated data regarding roles of NCLX in various kinds of cells, little is known about the roles in cardiomyocytes, where repetitive Ca 2þ transients occur and where a huge amount of ATP is generated and utilized. In the present study, we carried out a combination study of NCLX knockdown in spontaneously beating atrial cell line HL-1 and mathematical simulations. In HL-1 cells, NCLX knockdown by siRNA reduced the protein expression by~50%. A cytosolic Na þ-dependent mitochondrial Ca 2þ efflux was decelerated by knocking down NCLX, confirming that NCLX is a gene responsible for NCX mit in cardiomyocytes. Interestingly, the cycle length of spontaneous Ca 2þ oscillation and action potential generation was significantly prolonged by knocking down NCLX. Detailed inspection revealed that the rate of initial membrane depolarization and upstroke of Ca 2þ rise were markedly slower in the NCLX knockdown cells. Furthermore Ca 2þ content in sarcoplasmic reticulum (SR) was lower and SR Ca 2þ reuptake was slower in the NCLX knockdown cells. A mathematical model of HL-1 cells showed that an automaticity of HL-1 cells are driven by a spontaneous Ca 2þ leak from SR, called ''Ca 2þ clock''. Analyses using the model demonstrated that blocking NCX mit reduced SR Ca 2þ content and SR Ca 2þ leak, resulting in the prolongation of beating rate. Taken together, the NCLX has an important role in regulating cardiac automaticity by modulating ''Ca 2þ clock''.