The calcium pump sarcoplasmic reticulum Ca2+-ATPase (SERCA) counter-transports Ca2+ and H+ at the expense of ATP hydrolysis. SERCA uses separate proton and metal ion pathways during active transport to neutralize the highly charged transport site, thus preserving SERCA's structural stability during active Ca2+ transport. Although separate metal ion and proton pathways have been identified during slow (millisecond) structural transitions of SERCA, the existence of simultaneous metal and proton pathways during fast (microsecond) structural transitions remains unknown. We have analyzed microsecond-long trajectories of E1·H771+, a protonated intermediate of the pump populated during SERCA regulation. We found a transiently established hydrophobic pore in the luminal side of the transmembrane helices 6, 8, and 9. This narrow (0.5-0.6 nm) pore connects the transport sites to the sarcoplasmic reticulum lumen through a chain of water molecules. Protein pKa calculations of the transport site residues and structural analysis of the water molecules showed that this pore is suitable for proton transport. This transient proton pathway ensures neutralization of the transport sites during the rapid structural transitions associated with regulation of the pump. We conclude that this transient proton pathway plays a central role in optimizing active Ca2+ transport by SERCA. Our discovery provides insight into ion-exchange mechanisms through transient hydrophobic pores in P-type ATPases.
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© 2015 American Chemical Society.