Molecular mechanisms sensing and regulating physiological pH
My research interests focus on understanding the molecular physiology of solute transporters, channels, and signaling proteins involved in acid-base regulation and gas transport.
In my research studying the function of sodium-coupled bicarbonate cotransporters (NCBTs), in their roles regulating intracellular and whole-body pH and transepithelial transport I determine the extracellular pH (pHo) dependence, kinetics, and substrate specificity (HCO3− vs CO3=) of the NCBTs, e.g. NBCe1-A, and investigate which NCBT residues are responsible for imparting substrate specificity, electrogenicity, HCO3−-independent Na conductance. I employ two-electrode voltage clamp electrophysiology to record the currents and ion-sensitive microelectrodes to record the intracellular pH (pHi) or sodium concentration in cells heterologously expressing NCBTs. Out-of-equilibrium (OOE) solution perfusion exquisitely controls the extracellular environment of cells heterologously expressing NCBTs, allowing independent control of extracellular [CO2], [HCO3-] or pH.
My related acid-base physiology research focuses on the molecular processes underlying how the proximal tubule senses Δ[CO2]BL and Δ[HCO3-]BL and transduces these changes to modulate the rate of H+ secretion (JH) or bicarbonate reabsorption (JHCO3) during either respiratory or metabolic acidosis. Receptor protein tyrosine phosphatase γ (RPTPγ) is a major candidate for the CO2/HCO3- sensor. Changes in extracellular [CO2] and [HCO3-] result in changes in intracellular phosphatase activity. I use Förster resonance energy transfer (FRET) imaging to monitor the oligomerization or dissociation of RPTPγ as extracellular [CO2] or [HCO3-] changes in live cells, and also the how RPTPγ’s interactions with downstream signaling targets (e.g ErbB1 & ErbB2) changes as a consequence. Data recorded from samples exposed to in-equilibrium and OEE CO2/HCO3- solutions determines whether the highest RPTPγ phosphatase activity is activated by high CO2 or HCO3-, and which ligand is bound when RPTPγ is either a monomer or a dimer. The NCBTs are one of the major targets whose function is likely modulated by the activity of RPTPγ and its downstream signaling cascade.
I also study the movement of gas via protein channels (“gas channels”) across biological membranes. We developed the first assay for gas flux, based on maintaining the neutral buoyancy of a Xenopus oocyte previously injected with a 200-nl N2 bubble. During the neutral buoyancy assay (NBA) we apply pressure to the air above a saline solution, causing the bubble to constrict sufficiently that the oocyte falls to a depth of ~5 cm. As gas molecules dissolve in and diffuse through the cytoplasm, and eventually exit the cell, the bubble tends to collapse. A feedback system (camera, computer, digitally controlled pressure regulator) reacts by lowering the neutral-buoyancy air pressure (PNB) to maintain the oocyte at a 5-cm depth. The pressure inside the bubble (PBubble > PNB by a fixed amount) falls proportionally with the decreasing number of air molecules. The rate at which PNB falls thus reflects gas efflux from the bubble and can be converted into gas efflux in nmoles/s. We are also able to measure gas influx and can quantify N2, O2, or CO2 influx into an oocyte expressing NCBTs or other gas channel proteins (e.g. aquaporins or rhesus proteins) relative to control oocytes.
Molecular mechanism for sensing [CO2] and [HCO3−] by RPTPγ
HCO3− reabsorption (JHCO3) from renal proximal tubules (PT) is acutely regulated by basolateral [CO2] and [HCO3−], not by extracellular pH (pHo). More recently we reported that the knockout of receptor protein tyrosine phosphatase γ (RPTPγ), normally present in the PT basal membrane, eliminates the CO2 and HCO3− sensitivities of JHCO3, as well as the normal defense to whole-body metabolic acidosis (MAc). The RPTPγ intracellular region has both a D1 phosphatase domain and a D2 blocking domain. When RPTPγ dimerizes, the D2 domain of one monomer blocks the D1 domain of the other. The extracellular region possess a carbonic anhydrase (CA) like domain (CALD) that is strikingly similar to classic CAs. However, the CALD lacks the amino acid residues believed necessary for CA activity. If the CALD is no longer capable of interconverting CO2 and HCO3−, we hypothesize that it can sense CO2 or HCO3− and that the identity of the ligand bound to the CALD favors either dimerization or monomerization of the intracellular RPTPγ phosphatase domains. To detect the interaction of two RPTPγ monomers, we fuse the protein with the pH- and halide-insensitive GFP variants Aquamarine (Aq) to serve as a Förster resonance energy transfer (FRET) donor, and Citrine (Cit) to serve as a FRET acceptor, and coexpress the fusion proteins in HEK cells. We subject the cells to solutions representing different acid-base disturbances and acquire data from coexpressing cells, normalizing FRET for donor and acceptor expression levels (NFRET). We are able to vizualize changes in RPTPγ dimerization as reported by changes in the NFRET in response to each acid-base disturbance. In parallel experiments, we co-transfect cells with RPTPγ-Aq and ErbB1-Cit (a candidate downstream target for RPTPγ) and are able to measure changes in NFRET in response to acid-base disturbances that are reciprocal to those measured for RPTPγ dimerization. We hypothesize that HCO3− and CO2 compete at the CALD to control RPTPγ dimerization state (and presumably phosphatase activity). We also hyothesize that changes in the RPTPγ dimerization state in response to [CO2]o or [HCO3−]o but not pH, influence its interaction with downstream effector molecules including ErbB1.
Is NBCe1 an exchanger rather than a cotransporter?
I am recording the membrane potential (Vm), slope conductance and pHi from oocytes expressing NBCe1 that are exposed to different OOE solutions designed to probe the dependence of the transport direction on different extracellular ions and HCO3−.
Structural determinants of NBCe1 electrogenicity
To determine the structural elements of NBCe1 that are essential for electrogenicity, with emphasis on the contribution of extracellular loop 4.
The directional dependence of DIDS block of NCBTs
Identifying and examining the roles of extracellular DIDS binding sites and determining the directional dependence of DIDS block in NBCe1 and other Na+-coupled HCO3− transporters.
The role of carbonic anhydrase II on HCO3- -initiated transport through the SLC4A4 transporter NBCe1
Testing the metabolon hypothesis by investigating what influence, if any, of soluble carbonic anhydrase II (CAII) or CAII fused to the C-terminus of NBCe1A has on transporter slope conductance.
