I have two major areas of research interests:

1. Molecular mechanism underlying pain perception.
    Structure and function of Transient Receptor Potential (TRP) channels

2. Molecular mechanisms of action of centrally acting drugs.
    Structure and function of N-Methyl D-aspartate receptor (NMDAR) channels.

1. Molecular mechanism underlying pain perception.
    Structure and function of Transient Receptor Potential (TRP) channels
    A variety of TRP channels have been cloned. They are classified as TRPClassical, TRPVanilloid, TRPMelastatin, TRPAnkyrin and TRPPolycystin. TRP channels respond to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. Recent studies have shown that the role of TRP channels is much broader than sensory transduction. The research efforts will be dedicated to study these channels and determine their role in diseases. The structure and function of these receptors will be characterized the structure and function of these receptors using electrophysiological, immunohisto/cytochemical, molecular biological and biochemical techniques. The studies will be carried out at the level of singe molecule, single cell, whole organ and whole animal.
    TRPV1 is a well-characterized ion channel, which is activated by heat in the noxious temperature range (>42oC) and is critical for inflammatory thermal sensation. It is a Ca2+ permeant polymodal receptor activated by protons, anandamide, lipoxygenase metabolites of AA, N-arachidonyl dopamine, capsaicin (an active ingredient in hot chilli peppers) and resiniferatoxin (RTX, an ultrapotent agonist obtained from a cactus, Euphorbia resinifera) (Premkumar and Ahern, 2000; Premkumar et al., 2004; Premkumar and Raisinghani 2005). TRPV1 is distributed in the heart and blood vessels and is sensitized by prostaglandins (PG) via PKA and PKC mediated phosphorylation. Importantly, in the phosphorylated state, the activation threshold of TRPV1 is reduced below body temperature rendering the channel constitutively active. This might account for certain modalities of chronic pain conditions.
    Although, TRPV1 is primarily considered as a temperature sensor (>42oC), it is also distributed in regions that are not subjected to this temperature range. Activation of TRPV1 in sensory nerve endings supplying heart and blood vessels releases multiple vasoactive agents. Release of these agents from peripheral and central terminals of sensory neurons modulates nociceptive input from the periphery and synaptic transmission at the first sensory synapse, respectively. Moreover, localization of TRPV1 at the central terminals of the sensory neurons suggests that synaptic transmission at the first sensory synapse is likely to be modulated by activation or blockade of TRPV1. We propose that reduction of PG levels may contribute to deleterious vascular effects by decreasing sensitization of TRPV1 and subsequent reduction of CGRP and SP release. This possibility is supported by the finding that recovery from myocardial ischemia is compromised in TRPV1 knockout mice and proton mediated CGRP release from the heart is mediated exclusively by TRPV1. Since TRPV1 antagonists may become a part of the therapeutic armamentarium for painful conditions, it is imperative to determine whether blocking nociceptive receptors like TRPV1 decreases the release of vasoactive agents that are essential for homeostasis of the cardiovascular system.
    A serious effort is made to identify agonists and antagonists of TRPV1 for the treatment of chronic pain conditions. A number of analgesic balms available in the market contain capsaicin as the principal ingredient. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1) exhibits unique properties that can be exploited to treat chronic pain conditions. TRPV1, a Ca2+ permeable nonselective cation channel, is activated by physical and chemical stimuli and mediates inflammatory thermal sensation. Presently, this receptor is being considered as a target for analgesics through evaluation of different antagonists. A different approach is proposed since RTX is an agonist, which can achieve inhibition of nociceptive neurotransmission. RTX in picomolar concentrations cause slow, sustained, and irreversible activation of TRPV1. In the short term, the slow rate of depolarization allows the membrane potential to increment in a ramp-like fashion beyond threshold without generating action potentials (depolarization block), thus preventing pain during drug administration. In the long term, sustained irreversible activation increases intracellular Ca2+ leading to nerve terminal apoptosis, thus allowing long lasting pain relief.
    We are currently working on TRPV4, TRPA1 and TRPM8. TRPV4 and TRPA1 are mechanosensitive ion channels that are activated by osmolarity and tissue distention. On the other hand, TRPM8, a cold temperature sensor (activated between 24 and 10oC). We have found a reciprocal modulation of TRPV1 and TRPM8 by PKC (Premkumar et al., 2005). The implication of this finding is that there seems to be a balance between burning pain sensation and soothing cool sensation. PKC-mediated phosphorylation sensitizes TRPV1, but paradoxically, the cool/soothing sensation mediated by TRPM8 is blunted because of PKC-mediated dephosphorylation, leading to heightened pain perception. These findings could explain a novel mechanism by which inflammatory pain could be aggravated. Isoform-specific PKC inhibitors are in clinical trials, PKC? specific blockers have been proven to be useful in peripheral neuropathy. The pronounced effectiveness of this drug could be due to a dual effect of preventing TRPV1 sensitization and at the same time preserving TRPM8 function, which provides the much needed cool/soothing feeling in these conditions. Intriguingly, a receptor that is expressed in certain cancers, primarily prostate cancer, referred to as TRP-P8 is identical to that of TRPM8. Being a Ca2+ permeable channel, increases in intracellular Ca2+ levels can lead to apoptosis. Down regulation of TRPM8 and the subsequent reduction in the influx of Ca2+ may signal the cells to become antiapoptotic rather than proapoptotic, leading to cellular proliferation resulting in malignant growth.

2. Neurobiology of Neurotransmitter receptors
    Structure and function of N-Methyl D-aspartate receptor (NMDAR) channels.

    Neurotransmitters activate different kinds of receptors which mediate a variety of changes in ionic conductance. There have been extensive studies of conductance changes caused by glutamate, acetylcholine, nor adrenaline, dopamine, serotonin, GABA, opiates, adenosine and histamine. Most of these receptors are linked directly to ion channels, but some of these are coupled to channels via G-proteins, and others are coupled to enzyme systems which produce second messengers. Some of these receptors respond to stimulation by increasing intracellular calcium (For example, ionotropic (NMDA) and metabotropic glutamate receptors) and activation of calcium-dependent enzymes produces second messengers that can also modulate channel function. These ions channels are potential targets for modulation by physiological/-pharmacological agents.
    Since the cloning of the first NMDA receptor (NMDAR1) in 1991, eight different splice variants of NMDAR1, four different NMDAR2 (A-D) subunits and NMDAR-like (NMDAR-L, NR3A and B) subunit have been cloned. Although NMAR1 forms functional homomeric receptors in Xenopus oocytes, failure to produce functional channels in mammalian cell lines and the increase in current magnitude, when co-expressed with NMDAR2 subunit has indicated that the native NMDA receptors are probably heteromultimers. Channels formed by NMDAR1/NMDAR2A-D subunit combinations show differences in the properties with respect to their kinetics and modulation by physiological or pharmacological agents. The expression of these subunits is localized to specific regions of the brain and regulated developmentally. The NMDAR1 subunit is expressed throughout the brain at all ages. Infant death in transgenic mice lacking NMDAR1 subunits indicates the importance of this subunit. The differential expression of NMDAR2 subunits may impart specific properties to NMDA receptor channels. Presently, I am studying recombinant NMDA receptor-mediated ion channel function and its modulation by second messengers, phosphorylation, physiological and pharmacological agents. The NMDA receptor channel has been implicated in various important functions of the central nervous system due to its high Ca2+ permeability and the voltage-dependent Mg2+ block. I have already made significant progress in understanding Ca2+ permeability through the NMDA receptor channel. One of the mutants I studied revealed the presence of a high affinity divalent binding site at the entrance of the NMDA receptor channel. This binding site prefers calcium over magnesium (L. S. Premkumar and A. Auerbach, Neuron, 16: 869-880, 1996). For the first time, single channel noise analysis has been used to estimate the association, dissociation and permeation rate constants for Ca2+ and Mg2+. The observations that Na+ permeates when the Ca2+ is bound to the high affinity site, and is located at 0.1-0.2 through the membrane electric field from the external side indicate that the high affinity binding site is in the vestibule of the NMDA receptor channel. In my future experiments, I will address the following intriguing questions: What are the amino acid residues which comprise the putative Ca2+ binding site? What is the interaction of physiological and pharmacological agents on this site?

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