Targeting Sequence and Function-Dependence of Subcellular Localization of Transient Receptor Potential Mucolipin Channels

Wednesday, March 2, 2016 3025-Pos Board B402 Testosterone is a Highly Potent and Specific Agonist of TRPM8 Zahir Hussain1, Lusine Demirkhanyan2, Swapna Asuthkar2, Eleonora Zakharian2. 1 Department of Physiology, Umm Al-Qura University, Jeddah, Saudi Arabia, 2 Cancer Biology & Pharmacology, University of Illinois, Peoria, IL, USA. The role of the TRPM8 channel in the peripheral nervous system as a major receptor for cold temperatures has been well established. Less certainty was gained about the role of the channel beyond the nervous system. Particularly, the presence and high expression levels of TRPM8 in the prostate epithelium were not well understood. Recently, we identified that the prevalent male hormone, testosterone, is a highly potent endogenous agonist of TRPM8 channels. Picomolar concentrations of testosterone elicited rapid Ca2þ-uptake in cells expressing TRPM8, including prostate epithelial cells and neurons. Furthermore, testosterone evoked openings of the purified TRPM8 channel in planar lipid bilayers that were promptly inhibited with TRPM8 antagonists, or the purified androgen receptor (AR) protein. In the bilayers, TRPM8 activity was induced by both, permeable testosterone, as well as an impermeable analog, testosterone covalently conjugated to BSA. Interestingly, BSA-testosterone evoked openings of TRPM8 only into a small conductance state, with a mean slope conductance of ~7 pS, in comparison to ~37 pS obtained with the permeable analog. Furthermore, the prevalent female hormones, estradiol and progesterone, had no effect on the channel at picomolar or nanomolar concentrations, and only exerted openings at micromolar concentrations, but even then these steroids could open TRPM8 only into a small conductance state of ~8 pS. These results indicate that binding of steroids to the extracellular site induces conformational changes of the channel resulting in a narrow permeation path. Together, our results demonstrate that testosterone is not only a highly potent but also a highly specific agonist of TRPM8. 3026-Pos Board B403 Ubiquitin-Mediated TRPM8 Protein Degradation in the Pathogenesis of Prostate Cancer Swapna Asuthkar1, Alejandro Cohen2, Lusine Demirkhanyan1, Eleonora Zakharian1. 1 Cancer biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, USA, 2Proteomics and Mass Spectrometry Core Facility, Life Sciences Research Institute, Dalhousie University, Halifax, NS, Canada. Albeit TRPM8 mRNA is expressed at high levels, we found that TRPM8 protein undergoes ubiquitin-mediated degradation in prostate cancer (PC) cells. The cell lysates from five different PC cell lines showed 130 kDa protein band, corresponding to the molecular weight of TRPM8 along with additional lower molecular weight (LMW) bands migrating in the range of 100-55 kDa. The 55 kDa LMW band was most prominently seen in the androgenresponsive LNCaP cells. To delineate whether lower functional activation of TRPM8 protein in LNCaP cells could be mediated by TRPM8 degradation, we performed ubiquitination and mass spectrometry analysis. The UbiQaptureÔ-Q kit analysis showed increased capture of TRPM8 protein with anti-ubiquitin antibody. Furthermore, the mass-spectrometry analysis of a 130 kDa TRPM8 protein band, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of the initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. However, to further elucidate TRPM8 interacting network and to enrich the protein pool, we overexpressed myc-tagged TRPM8 in LNCaP cells. This strategy enabled us to use anti-Myc monoclonal antibody to precipitate TRPM8. When compared to untreated controls, the PYR-41/HF treated TRPM8 overexpressing LNCaP cells showed recovery of 130 kDa band detectible with coomassie blue staining. All the bands from each category were separately excised from the gel and estimated using tandem LC-MS/MS analysis. The TRPM8 precipitated from control cells mainly resulted in proteins involved in ubiquitination/ degradation. However, PYR-41/HF treated LNCaP cells not only recovered PMTRPM8 but also identified several apoptosis related proteins by MS analysis. We also found that activation of PMTRPM8 with drug treatment induced p53dependent-apoptotic mechanism that may play a protective role in PC progression through the regulation of rapid Ca2þ-influx. 3027-Pos Board B404 Competitive PIRT and PI(4,5)P2 Interactions Modulate TRPM8 Nicholas J. Sisco, Parthasarathi Rath, Wade D. Van Horn. School of Molecular Sciences, Arizona State University, Tempe, AZ, USA. Phosphoinositide regulator of TRP (PIRT) is a small human membrane protein that has been shown to modulate the function of specific TRP channels. PIRT has been implicated in fine-tuning the roles that TRP channels play in thermosensing, ligand activation, and pruritus. In an effort to better understand the role

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of PIRT in TRP channel modulation, PIRT has been expressed and purified in a biologically relevant state. Solution NMR studies of human PIRT have given way to backbone resonance assignment allowing for experimental determination of its membrane topology and residue specific characterization of PIRT interactions with the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and the TRPM8 sensing domain (helices S1-S4). PIRT contains a relatively unstructured N-terminus with two transmembrane helices followed by a positively charged PIP2 interacting site in the juxtamembrane region near the C-terminus. The binding affinity of PIRT to PI(4,5)P2 indicates the protein–lipid interaction is physiologically relevant. Interestingly, residues that specifically bind PI(4,5)P2 also specifically interact with the TRPM8 sensing domain. The overlap in PIRT binding sites between TRPM8 and PI(4,5)P2 suggests a lipid shuttling mechanism that could add an additional layer of regulation to TRPM8 function. These studies provide a structural and molecular framework to begin to dissect the regulatory mechanism of the tripartite PIRT–TRPM8–PI(4,5)P2 complex. 3028-Pos Board B405 Implications of Human TRPM8 Channel Gating from Sensing Domain and Menthol Binding Studies Parthasarathi Rath. School of Molecular Sciences, Arizona State University, TEMPE, AZ, USA. The human transient receptor potential melastatin 8 (hTRPM8) ion channel is a nonselective cation channel involved in human health and disease such as cancer, pain, obesity and diabetes. hTRPM8 has been shown to be gated in a polymodal manner by voltage, cold temperature, lipids, modulatory proteins, and chemical ligands. More specifically, hTRPM8 is activated by a number of cold inducing small molecules, including menthol and icilin. A better understanding of the molecular mechanism of ligand-gated channel activation may prove useful in unlocking the TRPM8 therapeutic potential. The sensing domain (transmembrane helices S1-S4) of TRPM8 has been implicated as the nexus of ligand binding, which is coupled to channel gating. Here, we present the direct binding of menthol to the isolated hTRPM8 sensing domain with nuclear magnetic resonance (NMR), circular dichroism (CD), and microscale thermophoresis (MST). We then compare the effects of binding to two previously identified residues, Y745 (on S2) and R842 (on S4), that have been shown to abrogate TRPM8 menthol sensitivity. The data presented indicate specific binding of menthol to the wild type as well as Y745H and R842H mutant hTRPM8 sensing domains, suggesting that menthol binding and the subsequent coupling to hTRPM8 channel gating are distinct processes. 3029-Pos Board B406 Biophysical Characterization of Human Transient Receptor Potential Melastatin 8 (TRPM8) Ion Channel Modulation by Phosphoinositide Regulator of TRP (PIRT) Jacob K. Hilton, Nicholas J. Sisco, Parthasarathi Rath, Wade D. VanHorn. School of Molecular Sciences, Arizona State University, Mesa, AZ, USA. Transient Receptor Potential Melastatin 8 (TRPM8) is the primary cold sensor in mammals. Besides thermosensation, this channel plays an important role in diverse physiological and pathophysiological processes. TRPM8 activity is modulated by various mechanisms, including via interaction with an accessory protein known as PIRT. Here, we present the first characterization of the human TRPM8-PIRT interaction. Using nuclear magnetic resonance (NMR) and biochemical studies, we show that PIRT binds specifically to the sensing domain (SD, transmembrane helices S1-S4) of TRPM8. Binding saturates as the TRPM8SD:PIRT mole ratio approaches 1, suggesting a 1:1 stoichiometry. Additionally, we performed comparative electrophysiology studies demonstrating that PIRT has a general attenuating effect on TRPM8 currents, in contrast to the potentiating effect observed in the mouse TRPM8-PIRT interaction. These results demonstrate the species dependent nature of TRPM8 modulation by PIRT and suggest a mechanism of tuning TRPM8 function across species. 3030-Pos Board B407 Targeting Sequence and Function-Dependence of Subcellular Localization of Transient Receptor Potential Mucolipin Channels Jian Xiong, Xinghua Feng, Michael X. Zhu. Integrative BIology and Pharmacology, UT Houston, Houston, TX, USA. Both of TRPML1 and TRPML3 are members of the mucolipin subfamily of Transient Receptor Potential (TRP) channels. They have been implicated in endolysosomal functions such as divalent cation release, luminal pH regulation and vesicle trafficking. Interestingly, whereas TRPML1 is almost extensively localized in lysosomes, TRPML3 is typically found in both lysosomes and the plasma membrane (PM). It has been shown that TRPML3 localization depends on TRPML1, but the mechanism remains unknown. Recently, a number of TRPML1 mutants have been reported to be either retained in the endoplasmic reticulum (ER) or preferentially targeted the PM. Coexpressing these

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Wednesday, March 2, 2016

mutants with either wild type TRPML3 or a non-functional ER-retained mutant, TRPML3-KK, we examined how differentially targeted TRPML1 proteins affect TRPML3 localization. We show that while the wild type TRPML1 decreased the PM targeting of TRPML3, the PM-targeted TRPML1 mutants did not enhance PM localization of TRPML3. Interestingly, not only the wild type TRPML1 brought TRPML3-KK to lysosomes, but also TRPML3 brought the ER-retained TRPML1-KK to these acidic organelles. Moreover, coexpression of TRPML-KK not only reduced TRPML3-mediated Ca2þ response to a TRPML agonist, but also nearly abolished PM localization of TRPML3. We conclude that while the lysosome targeting sequence(s) is important for sorting TRPML channels out of ER-Golgi network, the ion-conducting function may be critical for PM trafficking of these endolysosomal channels.

Myosins 3031-Pos Board B408 Structural Coordination of the Myosin Powerstroke Joseph Muretta, John Rohde, David D. Thomas. University of Minnesota, Minneapolis, MN, USA. Myosins drive actin-based motility required for important cellular functions and are model systems for enzyme based mechanochemical coupling. We are investigating the structural dynamics of myosins to determine how the movements of key structural elements are coordinated with the actin-activated ATPase cycling. We used FRET to investigate how the myosin powerstroke coordinates with actin-induced phosphate release in skeletal and cardiac myosins, two important myosin isoforms with disease relevance. In skeletal myosin, we find that weak actin binding triggers the myosin powerstroke before hydrolyzed phosphate dissociates from the myosin catalytic domain. We also find that the free energy change for the actin-induced light-chain domain rotation is small before phosphate is released, but large after it is released. This is consistent with a thermal diffusion model where the initiation of the myosin powerstroke is constrained by rapid diffusion of the light-chain domain between pre and post powerstroke orientations. In cardiac myosin, the kinetics of light-chain domain rotation and phosphate release are slower and not clearly resolved from each other. Interestingly, we find that addition of the cardiac myosin modulator omecamtiv mecarbil, accelerates actin-induced phosphate release, but slows the powerstroke. These results suggest that coordination of phosphate release and the myosin powerstroke is flexible, and that changing this coordination will change the force generating properties of myosin isoforms. Supported by: Graduate Excellence Fellowship-University of MN (JAR), NIH AR032961 & AR057220 (DDT), the Paul and Sheila Wellstone Muscular Dystrophy Center and American Heart Association (JMM). 3032-Pos Board B409 Mechanism of Cooperative Force Generations between Skeletal Myosins Motoshi Kaya1, Yoshiaki Tani1, Takumi Washio2, Toshiaki Hisada2, Hideo Higuchi1. 1 Physics, University of Tokyo, Tokyo, Japan, 2Frontier Science, University of Tokyo, Tokyo, Japan. To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosinrod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of power strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple power stroke states further enhance a chance of power stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of power stroke synchronization, since the strain-dependent transitions accompanied by the first or second power stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of power strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 mM ATP. These results were consistent with experimental results, supporting our conclusions. 3033-Pos Board B410 b-MYHC Mutations Linked to Early-Onset HCM and DCM Show Differences in Pre-Steady and Steady State Kinetic Parameters Carlos D. Vera Velazquez1, Jonathan Walklate2, Michael A. Geeves2, Leslie A. Leinwand1. 1 BioFrontiers Institute, University of Colorado-Boulder, Boulder, CO, USA, 2 School of Biosciences, University of Kent, Canterbury, United Kingdom.

Hypertrophic Cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young athletes and has an incidence of 1 in 500 people. Dilated cardiomyopathy (DCM) has a prevalence of 1 in 2000 people. Both diseases can be inherited, with HCM being autosomal dominant, and DCM having different genotypic profiles. There are over 400 mutations in cardiac myosin (MYH7) that have been linked to both diseases. Also noteworthy, early-onset HCM and DCM patients typically have a worse prognosis than adult-onset patients. The only intervention for these pediatric patients is a heart transplant. Here we describe the kinetic analysis of several cardiac myosin mutations that have been identified to be unique to early-onset patients. One set of mutations has been identified in pediatric HCM and the other set is DCM. Steady-state characterization of ATP hydrolysis for both mutant sets using an NADH coupled ATPase assay is reported. Additionally, transient kinetic analysis for several mutants was conducted illustrating differences in the cross-bridge kinetics between HCM and DCM mutants, namely ATP-induced dissociation, ADP release, and the binding constants of actin and the different nucleotide states. This data suggests that the motor activity is different among mutants and may provide insight into the mechanistic defects that can lead to both diseases. 3034-Pos Board B411 Arachidonic Acid Directly Binds and Activates Beta-Cardiac Myosin in the Regulated Cardiac Actomyosin Complex Manuel H. Taft1, Giulia Falorsi1,2, Michael B. Radke1, Salma Pathan-Chhatbar1, Nikolas Hundt1, Claudia Thiel1, Mirco Mu¨ller1, Vincenzo Lombardi2, Dietmar J. Manstein1. 1 Biophysical Chemistry, Hannover Medical School, Hannover, Germany, 2 Laboratory of Physiology, Department of Biology, Universita` di Firenze, Firenze, Italy. Mutation-induced dysfunction or misfolding of heart muscle sarcomere components have been linked to dilated and hypertrophic cardiomyopathies. We have previously shown that the small molecule EMD57033 increases b-cardiac myosin activity and acts as a pharmacological chaperone that stabilizes and refolds the motor domain. Following up on this finding, we investigated whether metabolites such as fatty acids can have a similar effect on b-cardiac myosin. Arachidonic acid (AA) was previously reported as an activator of smooth muscle myosin. We found that among all fatty acids tested, AA induced the largest effect on actin-activated ATPase activity of b-cardiac myosin. Closer characterization revealed a 13-fold increase in b-cardiac myosins affinity for actin in the presence of ATP and a 7-fold increase in the rate of phosphate release. We further aimed to elucidate the effect of AA-induced myosin activation in the context of a reconstituted human cardiac actin-troponin-tropomyosin complex. Both, assays monitoring ATPase and in vitro motility showed a significant increase in activity over a wide range of Ca2þ concentrations. Similar to the effect of EMD57033, the addition of AA increases the stability of myosin and reverses the loss of myosin activity caused by temperature stress. The refolding reaction can be monitored by the recovery of the intrinsic protein fluorescence signal that is associated with ATP binding and active turnover. Alternatively, we followed the clear reduction in the number of immobile filaments following the addition of AA. 3035-Pos Board B412 Modelling of Double Hit Mutations in Thoracic Aortic Aneurysm Disease that have Variable Impact on Phenotype Brett D. Hambly1, Elizabeth Robertson2, Stefanie S. Portelli1, Yaxin Lu1, Murat Kekic1, Richmond Jeremy2. 1 Pathology, University of Sydney, Sydney, Australia, 2Central Clinical School, University of Sydney, Sydney, Australia. Multiple genes are associated with thoracic aortic aneurysm (TAA) formation, including FBN1, TGFBR1&2, COL3A1 & 5A2, and MYH11. We hypothesized that given the population incidence of mutations in these genes it is likely that ‘double-hit’ mutations occur, which may result in an altered clinical phenotype when compared with the single gene mutation. Genomic DNA analysis of 16 genes implicated in TAA was performed on 11 patients with inherited TAA disease. Of the patients analysed, one patient (P1) was identified as having a mutation in both MYH11 (c.2517G.C (pW839C)) and FBN1 (splice donor site c.4210þ1G>A). Another patient (P2) was identified as having both COL5A2 (c.3794A>G (pD1265G)) and FBN1 (c.5861T>G (p.F1954C)). All mutations were predicted by PolyPhen to be deleterious. P1 has Marfan Syndrome (MFS), caused by the FBN1 mutation, and has severe aortic dilatation while his mother (M1), who also has MFS but does not have the MHY11 mutation, developed mild aortic dilatation, suggesting that the MHY11 mutation in P1 has resulted in a more aggressive phenotype. P2, siblings S1 and S2 and father (F2) all have relatively less severe MFS with progressive moderate dilatation of the aorta. Thus, the phenotype of pedigree 2 is similar for all affected members, but only P2 has a double-hit mutation,