Greater Cleveland
My research interests focus on understanding the molecular physiology of sodium-coupled bicarbonate cotransporters (NCBTs), aquaporins (AQPs), and Receptor Protein Tyrosine Phosphatases (RPTPs) in their role regulating intracellular and whole-body pH and transepithelial transport. I employ two-electrode voltage clamp electrophysiology to record the currents and ion-sensitive microelectrodes to record the intracellular pH or sodium concentration in cells heterologously expressing NCBTs. Delivery of out-of-equilibrium solutions allows exquisite control over the extracellular environment of cells heterologously expressing NCBTs in order to independently control extracellular [CO₂] and [HCO₃-] and pH. 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 or bicarbonate reabsorption during either respiratory or metabolic acidosis is another research focus. Receptor protein tyrosine phosphatase γ (RPTPᵧ) is a major candidate for the CO₂/HCO₃⁻ sensor. Förster resonance energy transfer imaging monitors the oligomerization of RPTPᵧ when [HCO₃⁻] or [CO₂] change, or when RPTPᵧ interacts with downstream signaling targets in live cells. I also investigate the function of several members of the AQP, rhesus (Rh) and solute transporter protein families, specifically with respect to their role as gas channels. I hope to make a significant contribution to the field by understanding the modulation, molecular composition and structural determinants of function in these transporter and sensor molecules relates to maintenance of intracellular and whole-body pH and control of transepithelial transport Specialties: Förster resonance energy transfer (FRET) and other imaging techniques Electrophysiology: Whole-cell patch clamp and two electrode voltage-clamp electrophysiology. Ion sensitive microelectrode fabrication and use. Out-of-equilibrium CO₂/HCO₃- solution perfusion. Molecular Biology & Protein Biochemistry: Diverse RT-PCR and cloning techniques; northern & western blot; In vitro transcription and translation techniques; Antibody purification; ELISA; immunocytochemistry. Microfluorimetry, Mammalian cell tissue culture, Flow cytometry.
In order to understand the molecular processes modulating the function of sodium-coupled bicarbonate cotransporters (NCBTs) in their roles regulating intracellular and whole-body pH and transepithelial transport, I use two-electrode voltage clamp electrophysiology to record the currents, ion-sensitive microelectrodes to record the intracellular pH (pHi) and sodium concentration in cells heterologously expressing NCBTs. Delivery of out-of-equilibrium (OOE) solutions allows exquisite control over the extracellular environment of cells heterologously expressing NCBTs in order to independently control extracellular carbon dioxide and bicarbonate concentrations and pH.
I lead a team of scientific moderators who maintain the site content and facilitate networking between the users of each forum.
As a volunteer moderator of the life science web site, www.ScientistSolutions.com I helped troubleshoot researchers’ experimental problems, find sources of the best equipment, protocols and literature and to form professional connections while maintaining the quality and integrity of the site's scientific content.
My research at Caltech had two concurrent projects. The first focused on understanding the density, intracellular processing, interactions, trafficking, and oligomerization of the GABA transporter mGAT1, by expressing fluorescent protein fusions in vitro. Understanding such processes in GAT1 and other related proteins is important for our understanding of their roles in diseases such as epilepsy, schizophrenia and depression. I developed a novel Förster Resonance Energy Transfer (FRET) technique that allows the oligomerization state of ion channels and transporters in specific subcellular compartments to be identified and which relates the FRET signature to function. We can now study the changes in oligomerization state/stoichiometry of different receptors and transporters in response to ligands, and how this correlates with their function and trafficking. This is particularly important for the understanding of the molecular mechanisms by which some diseases take effect and elucidating the events that underlie functional receptor up- or down-regulation in some addictions. In addition to my own GAT1 studies, I collaborated with my Lester lab colleagues to apply this FRET methodology to determine the changes in nicotinic acetylcholine receptor stoichiometry in response to chronic nicotine incubation. The second project was concerned with developing techniques to incorporate unnatural amino acids (UAAs) into ion channels expressed in mammalian cells. UAAs allow researchers to probe intermolecular interactions with a sensitivity unachievable by conventional mutagenesis. I applyied these techniques to incorporate UAAs at key positions in the pore of the hERG K+ channel when expressed in mammalian cells with the end-goal of improving drug safety by elucidating the subtle yet critically important nature of the binding interactions that determine how and why many non-cardiovascular clinical drugs bind to hERG and cause acquired LQT syndrome.
Performed patch clamp electrophysiology as part of collaborative hERG K+ channel pharmacology project between Neurion and Caltech.
Ph. D. Studentship in neuroscience with Prof. Annette Dolphin in the Dept. Pharmacology. The studentship was co-sponsored by the MRC and GlaxoSmithKline, so I spent a significant portion of my studies working at GSK in Stevenage, UK under the tutelage of Dr. Jeff Clare. I identified and cloned five human orthologs of the mouse stargazin gene, whose product was at the time hypothesised to be the first example of a neuronal voltage dependent calcium channel (VDCC) gamma subunit. I investigated the differential tissue distribution and sub-cellular localization of each ortholog. Furthermore, I studied their influence on the biophysical properties of different VDCCs when transiently co-expressed in Xenopus oocytes. In addition to my thesis, the work generated two peer-reviewed research articles.