Model-Structure, Mutagenesis and Functional Characteristics of the Human Transporter, NHA2

Human NHA2 is a novel member of the Cation/Proton Antiporters-2 (CPA2) family, linked to essential hypertension. Using the crystal structure of distant bacterial transporter NhaA as template, producing a model-structure of NHA2 necessitated a composite modeling approach. Through extensive mutagenesis guided by our model, we show that while NHA2 retained some functional and structural core elements of other Na+/H+ exchangers, it exhibited other significant exclusive features. A cluster of highly conserved titratable residues was located in the so-called assembly region, made of two discontinuous helices TM4 and TM11. Whereas in NhaA, oppositely charged residues have been proposed to compensate for partial dipoles generated within this assembly, we demonstrate that in NHA2 uncharged but polar residues suffice. Instead, NHA2 possesses unique, conserved charges predicted to interact with key essential residues. Combining structural data with evolutionary conservation analysis and mutagenesis, we propose a transport mechanism for NHA2, and compare it with mechanisms proposed for NhaA and NHE1. This study illustrates an attractive approach for studying new transporters, starting from structural data to guide initial experimental efforts.