The Transport Cycle of a Sodium/Proton Antiporter

Abstract

Sodium/proton antiporters, also known as sodium/proton exchangers, are essential secondary active transporters that are involved in pH homeostasis and regulation of sodium concentration and cell volume. They belong to the monovalent cation/proton antiporter families (CPA1 and CPA2) which all appear to share a common fold, spanning prokaryotic to mammalian family members. They obey general principles of secondary active transporters, namely they switch between at least two conformations in accordance with the alternating access model, the major states (outward facing (OF) and inward facing (OF)) are rooted in structurally breaking an inverted repeat symmetry, and they transduce energy from a driving ion gradient to vectorial substrate transport through conformational cycles. However, these broad principles constrain but do not immediately provide insights into the molecular mechanism of sodium/proton antiport. We have been using molecular dynamics simulations (long equilibrium MD, free energy calculations, enhanced sampling for rare events, constant pH simulations) in combination with experimental techniques such as X-ray crystallography and functional measurements to investigate key steps in the transport cycle of the prokaryotic CPA2 transporter TtNapA. A competitive binding mechanism between protons and sodium emerges from an analysis of the thermodynamics of the OF and IF states and naturally explains the experimentally observed activity peak in a narrow pH range. Direct computational sampling of the conformational transitions shows that NapA can undergo fast (sub-microsecond) conformational changes in either the proton or sodium-loaded states and reveals a distinct meta-stable intermediate state. Taken together, the information from all the major steps in the complete transport cycle can then form the basis for multi-scale kinetic models of the physiological function of the antiporter.