Changes in Temperature Have Opposing Effects on Current Amplitude in a7 and a4b2 Nicotinic

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric a7 nAChR, the more slowly desensitizing heteromeric a4b2 nAChR and on a7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21uC) were compared to those recorded at either physiological temperature (37uC) or at lower temperature (4uC). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with a4b2 nAChRs was increased at high temperature (12569%, n =6, P,0.01) and reduced at low temperature (4765%, n =6, P,0.01), whereas the amplitude of a7 responses was reduced at high temperature (2767%, n = 11, P,0.001) and increased at low temperatures (224616%, n = 10, P,0.001). In contrast to the effects of temperature on a4b2 and wild type a7 nAChRs, the amplitude of a7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when a7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonistevoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of recepto

NEUROCHEMISTRY NEUROREPORT

This study determined the role of ventral tegmental area acetylcholine and glutamate receptors in modulating laterodorsal tegmentum stimulation-evoked dopamine e¥ux in the nucleus accumbens. Rapid changes in dopamine oxidation current were measured at carbon ¢ber microelectrodes using ¢xed potential amperometry in urethane anesthetized male mice. Intraventral tegmental area infusions of the muscarinic acetylcholine receptor antagonist scopolamine, the nicotinic acetylcholine receptor antagonist mecamylamine, or the ionotropic glutamate receptor antagonist kynurenate signi¢cantly diminished dopamine e¥ux in the nucleus accumbens evoked by brief electrical stimulation of the laterodorsal tegmentum. These ¢ndings suggest that acetylcholine and ionotropic glutamate receptors in£uence rapid dopaminergic activity and thus the communication of behaviorally relevant information from ventral tegmental area dopamine cells to forebrain areas. NeuroReport 19:991^995 c 2008 Wolter

Slater's permanent address is Muscular Dystrophy Group Lab, Newcastle General Hospital,

ABSTRACT If skeletal muscles are damaged in ways that spare the basal lamina sheaths of themuscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites i

Nonstereoselective Inhibition of Neuronal Nicotinic

We have found that racemic ketamine strongly inhibits the current mediated through neuronal nicotinic acetylcholine receptors (nAchRs) in PC12 cells, a rat pheochromocytoma cell line. Ketamine stereoisomers have different potencies for the anesthetic action, with the S-enantiomer being about 3 times as potent as the R-enantiomer. The purpose of this study was to clarify if the inhibitory effects of ketamine on neuronal nAchRs contribute to their anesthetic effect. We compared potencies of ketamine enantiomers for neuronal nAchR inhibition with those for the anesthetic action. S(�) and R(�) ketamine inhibited the nicotine-induced whole-cell current in a dosedependent manner at the membrane potential of �60 mV. They accelerated the current decay, resulting i

The American Society for Biochemistry and Molecular Biology.

Nicotinic acetylcholine receptors (nAChR) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter acetylcholine (ACh) as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs have been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Our lab has reported that the muscle-type, α4β2, and α7 receptors bind agonist molecules via a cation-π interaction. 1-3 This chapter describes our efforts to elucidate the agonist binding mechanism of the α4β4 receptor. Unnatural amino acid mutagenesis and chimeric β subunits were used to probe the respective contributions of the α4β4 principal and complementary binding components to agonist binding and receptor pharmacology. Here, we report that the α4β4 receptor utilizes a strong cation-π interaction to

Both CY- and,&subunits Contribute to the Agonist Sensitivity of

A family of genes has been identified that encodes subunits of nicotinic acetylcholine receptors (nAChRs) and is expressed in the nervous system. Functional neuronal nAChRs can be expressed in Xenopus oocytes by injection of RNA encoding 1 of 2 different &subunits (/32,84) in pairwise combination with RNA encoding 1 of 3 different a-subunits (a2, (~3, a4). We examined the sensitivity of these 6 different a-B-subunit combinations to the nicotinic agonists ACh, nicotine, cytisine, and 1,l-dimethyl-4-phenylpiperazinium (DMPP). Each subunit combination displayed a distinct pattern of sensitivity to these 4 agonists. The a2B2 combination was B-fold more sensitive to nicotine than to acetylcholine, while the a382 combination was 17-fold less sensitive to nicotine than to ACh, and the a384 combination was equally sensitive to both nicotine and ACh. nAChRs composed o

In memory of my grandparents:

The struggle itself towards the heights is enough to fill a man’s heart. One must imagine Sisyphus happy. Albert Camus My six years of pushing the stubborn rock of graduate school uphill have come to an end. Thankfully, I’ve had many sets of hands helping me shove. The first set belongs to my advisor, Dennis Dougherty. In my senior year of college, while I was pondering graduate school, Dennis gave two lectures at M.I.T. I attended both and was hooked by his discussions of unnatural amino acids, cation-π interactions, and the brain. His ability to tell engaging scientific stories and to explain his reasoning in a transparent manner continues to inspire me. As an advisor, Dennis has allowed me a great deal of freedom, while also injecting timely doses of guidance. He has stood by me when projects appeared mired in confusion. And he has provided me the space to explore a non-traditional career path. For his support, academic and otherwise, I thank him. All of the research contained in the following pages would have been difficult to perform without the expertise of Henry Lester and his lab. Henry has served as a fresh set of eyes for perplexing data and a source of new experiment ideas during countless “Unnaturals Club ” meetings. I also greatly appreciate his support of my ventures into science writing. Bruce Cohen, a member of the Lester Group, has also been an excellent electrophysiology tutor. v I also thank the other members of my committee, Peter Dervan, Bill Goddard, and Dave Tirrell, for their words of encouragement and advice throughout my graduate career. When I arrived at CalTech I met an amazing collection of scientists in the Dougherty Group who made me feel truly welcome. The Lake Avenue lunch squad o

doi:10.1155/2012/103120 Review Article Pathogenesis of Abdominal Aortic Aneurysms:

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Inflammation, proteolysis, smooth muscle cell apoptosis, and angiogenesis have been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs), although the well-defined initiating mechanism is not fully understood. Matrix metalloproteinases (MMPs) such as MMP-2 and-9 and other proteinases degrading elastin and extracellular matrix are the critical pathogenesis of AAAs. Among the risk factors of AAAs, cigarette smoking is an irrefutable one. Cigarette smoke is practically involved in various aspects of the AAA pathogenesis. Nicotine, a major alkaloid in tobacco leaves and a primary component in cigarette smoke, can stimulate the MMPs expression by vascular SMCs, endothelial cells, and inflammatory cells in vascular wall and induce angiogenesis in the aneurysmal tissues. However, for the inflammatory and apoptotic processes in the pathogenesis of AAAs, nicotine seems to be moving in just the opposite direction. Additionally, the effects of nicotine are probably dose dependent or associated with the exposure duration and may be partly exerted by its receptors—nicotinic acetylcholine receptors (nAChRs). In this paper, we will mainly discuss the pathogenesis of AAAs involving inflammation, proteolysis, smooth muscle cell apoptosis and angiogenesis, and the roles of nicotine and nAChRs. 1

Biphasic, Opposing Modulation of Cloned Neuronal �1E Ca Channels by Distinct Signaling Pathways Coupled to M2

Neuronal �1E subunits are thought to form R-type Ca channels. When expressed in human embryonic kidney cells with M2 muscarinic acetylcholine receptors, Ca channels encoded by rabbit �1E exhibit striking biphasic modulation. Receptor activation first produces rapid inhibition of current amplitude and activation rate. However, in the continued presence of agonist, �1E currents subsequently increase. Kinetic slowing persists during this secondary stimulation phase. After receptor deactivation, kinetic slowing is quickly relieved, and current amplitude over-recovers before returning toward control levels. These features indicate that inhibition and stimulation of �1E are separate processes, with stimulation superimposed on inhibition. Pertussis toxin eliminates inhibition without affecting stimulation, demonstrating that inhibition and stimulation involve distinct signaling pathways. Neither inhibition nor stimulatio

Single Channel Studies of Inward Rectifier Potassium Channel Regulation by

abstract Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I KACh) by G protein–coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G � � subunits to the NH 2- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I KACh. Because of the very low basal activity of native I KACh, it has been difficult to determine the single channel effect of G � � subunit binding on I KACh activity. Through analysis of a novel G protein–activated chimeric inward rectifier channel that displays increased basal activity relative to I KACh, we find that single channel activation can be explained by a G protein–dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state. key words: ion channel • patch-clamp • G protein • gating • acetylcholin