Nazih L. Nakhoul, Ph.D.

My research is focused on studying the cellular and molecular mechanisms that regulate acid-base balance and intracellular pH particularly those relevant to renal epithelia. These studies involve using basic molecular biology techniques to express transporters and/or channels in frog oocytes and other mammalian cells in order to study the properties of these transport mechanisms. The physiological methods we have been using include ion-selective microelectrodes, two-electrode voltage clamp, optical measurements of intracellular pH by fluorescence and the isolated perfused kidney tubule preparation. Several projects are underway in my laboratory.

One of the most important studies we are currently undertaking include molecular and functional characterization of novel NH4+ (and likely NH3) transporters previously unidentified in the kidney. Renal NH3/NH4+ transport is extremely important for acid-base homeostasis and NH3 transport has been reported to act as an important signaling molecule that can modulate transport by other renal mechanisms such as Cl-HCO3- exchange or possibly Na+/(HCO3-)n . We isolated two clones from the kidney cortex, originally cloned from yeast. These non-erythrocyte membrane proteins belong to the Rh antigen family and are expressed in the cortical collecting duct, a site where NH3/NH4+ transport plays a crucial role. We have expressed these clones (known as Rhbg and Rhcg) in oocytes and our data show fundamental differences in how NH3/NH4+ are transported by the two Rh proteins. These studies indicate that these proteins have an unusual dual purpose of transporting a fixed ion (NH4+) and its volatile complement (NH3), two components of an important system essential for acid-base homeostasis. These studies are very novel in view of the fact that these transporters could be the first to act as specific NH4+ transporters and have never been characterized before.

We are also investigating CO2 transport, generally thought to cross membranes by solubility diffusion, and the possibility that it could be transported through water channels known as AQPs or other membrane proteins. This work, demonstrating that AQP1 is permeable to CO2, was the first evidence that a gas such as CO2 could be transported through a channel. As a continuation of this work, we are investigating the role of carbonic anhydrase in mediating CO2 transport. We have characterized the role of two isozymes, CA-II and CA-IV and we are currently investigating the interaction of CA-II and CA-IV with expressed acid-base transporters including the Rh proteins and the anion exchanger AE-1.

Other studies in the lab investigate structure-function characteristics of Rh proteins and aim at characterizing a molecular model that explains the ability of these molecules to transport ions and serve as gas channels as well. We are determining fundamental characteristics of Rh proteins in terms of substrate specificity, ion-selectivity and pH dependence. These studies are extremely important to understand how the distal nephron achieves transepithelial NH3/NH4+ transport to maintain systemic acid-base balance.

Laboratory Members:

• Tolga Caner – Graduate Student