Eric B. Gonzales

Inhibition of type a GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipines effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.

Stoichiometric analysis of the TM2 6′ phenylalanine mutation on desensitization in α1β2 and α1β2γ2 GABAA receptors

Abstract The presence of phenylalanine (F) at the 6′ position of transmembrane domain 2 (TM2) in the α4 subunit of α4β2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6′F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6′ position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6′ position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.

A review of creatine supplementation in age-related diseases: more than a supplement for athletes

Creatine is an endogenous compound synthesized from arginine, glycine and methionine. This dietary supplement can be acquired from food sources such as meat and fish, along with athlete supplement powders. Since the majority of creatine is stored in skeletal muscle, dietary creatine supplementation has traditionally been important for athletes and bodybuilders to increase the power, strength, and mass of the skeletal muscle. However, new uses for creatine have emerged suggesting that it may be important in preventing or delaying the onset of neurodegenerative diseases associated with aging. On average, 30% of muscle mass is lost by age 80, while muscular weakness remains a vital cause for loss of independence in the elderly population. In light of these new roles of creatine, the dietary supplement’s usage has been studied to determine its efficacy in treating congestive heart failure, gyrate atrophy, insulin insensitivity, cancer, and high cholesterol. In relation to the brain, creatine has been shown to have antioxidant properties, reduce mental fatigue, protect the brain from neurotoxicity, and improve facets/components of neurological disorders like depression and bipolar disorder. The combination of these benefits has made creatine a leading candidate in the fight against age-related diseases, such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, long-term memory impairments associated with the progression of Alzheimer’s disease, and stroke. In this review, we explore the normal mechanisms by which creatine is produced and its necessary physiology, while paying special attention to the importance of creatine supplementation in improving diseases and disorders associated with brain aging and outlining the clinical trials involving creatine to treat these diseases.

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.

Acidity and Acid-Sensing Ion Channels in the Normal and Alzheimer's Disease Brain.

Alzheimers disease prevalence has reached epidemic proportion with very few treatment options, which are associated with a multitude of side effects. A potential avenue of research for new therapies are protons, and their associated receptor: acid-sensing ion channels (ASIC). Protons are often overlooked neurotransmitters, and proton-gated currents have been identified in the brain. Furthermore, ASICs have been determined to be crucial for proper brain function. While there is more work to be done, this review is intended to highlight protons as neurotransmitters and their role along with the role of ASICs within physiological functioning of the brain. We will also cover the pathophysiological associations between ASICs and modulators of ASICs. Finally, this review will sum up how the studies of protons, ASICs and their modulators may generate new therapeutic molecules for Alzheimers disease and other neurodegenerative diseases.

Detergent screening of the human voltage‐gated proton channel using fluorescence‐detection size‐exclusion chromatography

The human voltage‐gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino‐ and carboxy‐termini. The protein is activated by membrane depolarization, similar to other voltage‐sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three‐dimensional structures for homologues of this voltage‐gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino‐terminus of the human Hv1 proton channel (termed EGFP‐Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence‐detection size‐exclusion chromatography. The EGFP‐Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3–12 and the nonionic n‐dodecyl‐β‐d‐maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size‐exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full‐length human Hv1 proton channel.

Identification of a unique Ca 2+ -binding site in rat acid-sensing ion channel 3

Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca2+) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca2+ results in channel opening, with the site’s location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.Acid-sensing ion channels (ASICs) sense changes in extracellular acidity with Ca2+ as an allosteric modulator and channel blocker. Here authors use electrophysiology and molecular dynamics simulation to identify the residue in ASIC3 which modulates proton sensitivity and contributes to the Ca2+ block.

Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

Targeted Acid-Sensing Ion Channel Therapies for Migraine

Acid-sensing ion channels (ASICs) are a family of ion channels, consisting of four members; ASIC1 to 4. These channels are sensitive to changes in pH and are expressed throughout the central and peripheral nervous systems—including brain, spinal cord, and sensory ganglia. They have been implicated in a number of neurological conditions such as stroke and cerebral ischemia, traumatic brain injury, and epilepsy, and more recently in migraine. Their expression within areas of interest in the brain in migraine, such as the hypothalamus and PAG, their demonstrated involvement in preclinical models of meningeal afferent signaling, and their role in cortical spreading depression (the electrophysiological correlate of migraine aura), has enhanced research interest into these channels as potential therapeutic targets in migraine. Migraine is a disorder with a paucity of both acute and preventive therapies available, in which at best 50% of patients respond to available medications, and these medications often have intolerable side effects. There is therefore a great need for therapeutic development for this disabling condition. This review will summarize the understanding of the structure and CNS expression of ASICs, the mechanisms for their potential role in nociception, recent work in migraine, and areas for future research and drug development.

Effects of creatine supplementation on nociception in young male and female mice

BACKGROUND The objective of this study was to evaluate creatine as an anti-nociceptive compound in an animal model of thermal and inflammatory pain. Creatine has the structural potential to interact with acid-sensing ion channels (ASIC), which have been involved in pain sensation modulation. The hypothesis evaluated in this study was that creatine will interact with ASICs leading to decreased nociception. METHODS Male and female C57BL/6J mice were fed with either a control diet or the control diet supplemented with creatine (6.25 g/kg diet). After one week on the diet, the mice were tested for thermal hyperalgesia and inflammatory pain response. RESULTS The latency to withdraw the tail during the thermal hyperalgesia test was unaffected by sex or diet. During the formalin test, males and females responded differently to the stimulus, and the female mice supplemented with creatine seemed to recover faster than the controls. To determine whether ASICs mediate the action of creatine, GMQ, an ASIC3 agonist, was injected in one paw and pain response was quantified. Females responded more strongly to GMQ injections, and all mice fed creatine had a decreased response to GMQ. CONCLUSIONS These preliminary data suggest a potential effect of creatine on inflammation-based nociception that may be mediated via ASIC3. While preliminary, this study warrants further research on the potential of creatine as an analgesic and can serve as a stepping stone for the development of ASIC-based therapeutics.