Is ASP-454 Necessary for Na+ and K+ Binding in the Glutamate Transporter EAAC1?

Abstract

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''.

Transient Kinetics Reveal Mechanism and Voltage Dependence of Inhibitor and Substrate Binding to Glutamate Transporters

“…To further test our MD simulation results with a system containing aspartate as the ligand, for which force field parameters are well-established, we performed simulations using a EAAT3 homology model. 41 Simulations were performed without Na + occupying the Na2 site, which promotes opening of hairpin loop 2 and destabilizes the bound amino acid. 42,43 As shown in Figures 4A and B, aspartate was stable for up to 760 ns in the binding site at 0 mV.…”