Stephen R. Ikeda, MD, PhD - US grants

The aim of the proposed research is to further our understanding of the role of the sympathetic neuron calcium current in the pathogenesis and maintenance of hypertension. Recent studies have shown that an exaggerated release of norepinephrine (NE) from peripheral adrenergic nerve terminals may e one of the mechanisms producing hypertension in the spontaneously hypertensive rat (SHR) model of primary hypertensin. Although the mechanism underlying this phenomenon remains obscure, it has been proposed that a dysfunction in the presynaptic receptor mediated regulation of calcium-dependent exocytotic NE release may be responsible. Thus, the hypotheses to be tested are that: 1) an alteration in the biophysical properties and/or 2) an alteration in the neurotransmitter modulation, of the sympathetic ganglion voltage-gated calcium current is associated with the development and/or maintenance of hypertension in SHR. Accordingly, the Specific Aims of the proposal are to compare the properties ad neurotransmitter modulation of the voltage-gated calcium current of superior cervical ganglion (SCG) neurons acutely isolated from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats. This will be accomplished using the patch-clamp technique in the whole-cell voltage- clamp mode. The following calcium current processes will be studied: 1) biophysical properties, i.e. activation, dihyrodopyridine sensitivity, and current density; and 2) the effect of and concentration-response relationship for NE and adenosine, agents which inhibit NE release, and isoproterenol and angiotensin II, agents which stimulate NE release. These studies will be done on both young SGR (4-6 weeks) in the "prehypertensive phase" ad older SHR (20-24 weeks) in the "established phase" of hypertension to explore the relationship between development of hypertensin and calcium current properties. In addition, studies will be performed to determine whether calcium current alterations are specific to the genetic SHR model of hypertensin. Although there is considerable evidence suggesting a role for the peripheral sympathetic nervous system in the pathogenesis and maintenance of hypertension in SHR, the electrophysiology of sympathetic ganglion neurons in SHR has received little attention. It is hoped that the proposed study will help fill this void in our understanding of the pathobiology of hypertension and provide a basis for the development of improved antihypertensive agents.

Voltage-gated Ca2plus channels of the N-type are modulated via a number of discrete signaling pathways. The most commonly utilized pathway involves a membrane-delimited mechanism whereby receptor activation of a pertussis toxin-sensitive heterotrimeric G protein results in a distinct form of voltage-dependent inhibition. Despite intense investigation, the molecular mechanism underlying this signal transduction pathway is at present unknown. Thus, the long term goal of our research is to further our understanding of the molecular mechanisms underlying modulation of N-type Ca2plus channels. The main hypothesis to be tested is that neurotransmitter modulation of N-type Ca2plus channels is mediated by specific G-protein beta gamma-subunits which bind directly to a site on the Ca2plus channel photo1B-subunit to produce voltage- dependent channel inhibition. A combination of electrophysiological, molecular biological, and biochemical methods will be used to investigate modulation of both native and heterologously expressed N- type Ca2plus channels. Accordingly, the SPECIFIC AIMS are: 1) to identify the G protein subunit, i.e., Gphoto or G beta gamma, which mediates voltage-dependent N-type Ca2plus channel modulation; 2) to determine whether G protein subunit composition confers specificity in regard to voltage-dependent N-type Ca2plus channel modulation and receptor coupling; 3) to determine the site on the Ca2plus channel involved in G beta gamma subunit binding. These experiments will generate new information regarding the molecular mechanism of voltage-dependent N-type Ca2plus channel inhibition. The elucidation of this mechanism has broad implications as numerous G protein-coupled receptors, any of which are implicated in disease or are targets of therapeutics agents, are thought to modulate synaptic transmission in the peripheral and central nervous system, at least in part, via this mechanism.

Metabotropic glutamate receptors (mGluRs) compromise a unique subfamily of G-protein couple receptors for which glutamate is the endogenous ligand. A number of physiological roles have been ascribed to mGluRs including ion channel modulation, regulation of neurotransmitter release, and participation in synaptic plasticity such as long-term potentiation and depression. Moreover, recent evidence suggests that mGluRs could play a role in pathological processes related to epilepsy, ischemia, excitotoxic neurodegenerative disorders, Alzheimer's Disease, and hypertension. Thus there is a substantial interest in defining the roles subserved by specific mGluR subtypes and developing subtype specific pharmacological agents. At present, the sequences for twelve mGluR subtypes (mGluR1-8 plus splice variants) have been elucidated. However, despite this wealth of primary sequence information, little is known about which specific mGluR subtypes modulate ion channels and synaptic transmission in neurons or the mechanisms involved in these actions. To address this void in our knowledge, we will examine the function, pharmacology, and signal transduction pathways of: 1) heterologously expressed mGluRs in sympathetic neurons (SPECIFIC AIMS 1 and 2), and 2) natively expressed mGluRs in CNS neurons (SPECIFIC AIMS 3 and 4). Accordingly, the SPECIFIC AIMS are: 1) Pharmacological characterization and signal transduction pathways of defined mGluR subtypes that inhibit N-type Ca2+ and M-type K+ channels. Molecularly defined mGluR subtypes will be heterologously expressed in mature rat sympathetic neurons to determine which subtypes modulate N-type Ca2+ and M-type K+ channels. 2) Molecular elements of mGluR structure/function. The structural domains of mGluRs which determine pharmacological profile and G-protein coupling specificity will be examined by expressing chimeric mGluR constructs in sympathetic neurons. 3) Identification and signal transduction pathways of mGluRs involved in Ca2+ and K+ channel modulation in isolated CNS neurons. The pharmacological (agonist and antagonist) profile and signal transduction characteristics of mGluR-mediated Ca2+ and K+ channel inhibition will be determined in acutely isolated hippocampal and cortical neurons. 4) Identification of mGluRs involved in modulation of synaptic transmission in CNS neurons. Glutamatergic synaptic transmission in brain slices will be examined to determine which mGluRs mediate modulate neurotransmitter release.

The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. A recently completed project provides the first detailed description of gene structure and sequence for the human gene FFAR3 (GPR41) and an adjacent duplicated gene, GPR42. FFAR3 is a G-protein coupled receptor for which short-chain fatty acids (acetate, propionate, and butyrate) serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic β-cells, and sympathetic neurons and has been implicated in a number of conditions including obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is part of a tandem segmental duplication that results in a duplicon termed GPR42 that shares > 99% sequence identity with FFAR3. The high sequence identity renders short-read sequencing of FFAR3 and GPR42 unreliable. Moreover, GPR42 is classified as a suspected pseudogene based on lack of function when heterologously expressed. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (20%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the individual's GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons uncovered a variety of pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors. Puhl HL III, Won Y-J, Lu VB, Van B Lu, Ikeda SR. 2015. Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed. Sci Rep 5: 12880. A second completed project described the expression pattern of voltage-gated sodium channel, Nav1.8 (encoded by the Scn10a gene). Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP- positive (mean diameter = 26.5 um). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (approximately 10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8- immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons. Lu VB, Ikeda SR, Puhl HL. 2015. A 3.7 kb fragment of the mouse SCN10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal. J Neurosci 35: 80218034. In addition to these projects, two collaborative manuscripts were published: 1) Iyer MR, Cinar R, Liu J, Godlewski G, Szanda G, Puhl H, Ikeda SR, Deschamps J, Lee Y-S, Steinbach PJ, et al. 2015. Structural basis of species-dependent differential affinity of 6-alkoxy-5-aryl-3-pyridinecarboxamide cannabinoid-1 receptor antagonists. Mol Pharmacol 88: 238244. We provided cannabinoid receptor mutants used in this study which exposed pharmacological differences between rodent (rat and mouse) and human receptors. 2) Nguyen TA, Sarkar P, Veetil JV, Davis KA, Puhl HL, Vogel SS. 2015. Covert changes in CAMKII holoenzyme structure identified for activation and subsequent interactions. Biophys J 108: 21582170. Dr. Henry Puhl provided training and advice for generating CamKII constructs used in this study of enzyme structure function using optical techniques.