| Na+/H+ exchangers (NHE) are transmembrane proteins that mediate the electroneutral exchange of Na+ and proton across the plasma membrane. There are seven mammalian isoforms of the gene family identified to date. The recently cloned isoform NHE5 seems to be the most highly cell type-specific being probably expressed only in neurons. The objective of my research was to describe the basic biochemical and some of the regulatory characteristics of NHE5 in an effort to understand its in vivo function. To this end, the full-length cDNA of NHE5 was reconstructed and transfected into the NHE-deficient Chinese hamster ovary cell line AP1.;Pharmacological analyses demonstrated that H+ i-activated 22Na+ influx mediated by NHE5 was inhibited by several classes of drugs at half-maximal concentrations that were intermediate to those determined for the high-affinity NHE1 and the low-affinity NHE3 isoforms. Kinetic analyses showed that the extracellular Na+-dependence of NHE5 activity followed a simple hyperbolic relationship and, unlike other NHE isoforms, the intracellular H+-dependence also exhibited first-order kinetics. Extracellular monovalent cations, such as H+ and Li+, but not K+, acted as effective competitive inhibitors of 22Na+ influx by NHE5.;To find novel interacting proteins that are involved in NHE5 regulation, a yeast two-hybrid screen of human brain cDNA library was conducted using NHE5 as bait. A clone encoding the AMP-activated protein kinase (AMPK) alpha2 subunit was further analyzed. AMPK is a serine/threonine kinase that is activated by elevated ratios of [AMP]/[ATP], regulating various biological processes in response to hypoxia or exercise. AMPK alpha2 binds NHE5 in vitro and in vivo, and directly phosphorylates it in vitro. Activation of endogenous AMPK by AICAR, a membrane permeable AMP analogue, as well as heterologous expression of the full-length and constitutive active forms of alpha2 subunit increased the transporter activity measured by 22Na+ influx.;The regulatory protein arrestin3 was also found to interact with NHE5 in the yeast two-hybrid screen. Arrestins were previously shown to associate with and regulate transmembrane proteins of the G protein-coupled receptor family. We demonstrate that NHE5 binds arrestin3 both in vitro and in vivo; and the binding is phosphorylation-dependent. When co-expressed in CHO cells, arrestin3 and NHE5 co-localize, and arrestin3 expression seems to attenuate the basal activity of the transporter. The data presented in this thesis reveals new aspects of both NHE regulation, and AMPK and arrestin function. |
