DOCSLIB.ORG

Mutations at Two Distinct Sites Within the Channel Domain M2 Alter Calcium Permeability of Neuronal A7 Nicotinic Receptor D

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mutations at Two Distinct Sites Within the Channel Domain M2 Alter Calcium Permeability of Neuronal A7 Nicotinic Receptor D Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6971-6975, August 1993 Neurobiology Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal a7 nicotinic receptor D. BERTRANDt, J. L. GALZI*, A. DEVILLERS-THIERY*, S. BERTRANDt, AND J. P. CHANGEUXt tNeurobiologie Mol6culaire, Unit6 de Recherche Associ6e au Centre National de la Recherche Scientifique D1284, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France; and tD6partement de Physiologie, Centre Medical Universitaire (Faculte de Medecine), 1211 Geneva 4, Switzerland Contributed by J. P. Changeux, April 2, 1993 ABSTRACT The relative permeability for sodium, potas- (E237A), indirect evidence suggested significant alterations sium, and calcium of chicken a7 neuronal nicotinic receptor of Ca2+ permeability (14). was investigated by mutagenesis of the channel domain M2. In this study, we further investigate the monovalent vs. Mutations in the "intermediate ring" of negatively charged divalent cation selectivity of the a7 receptor. We show that residues, located at the cytoplasmic end of M2 (site 1), reduce a mutation at the cytoplasmic end of M2 (E237A) abolishes calcium permeability without significantly modifying other Ca2+ permeability without significantly affecting other prop- functional properties (activation and desensitization) of the erties of the pharmacological and physiological responses to receptor; a similar change of ion selectivity is also noticed when ACh. We further identify another site of two adjacent amino mutations at site 1 are done in the context of a receptor mutant acids, close to the extracellular end of M2 (Leu-254 and that conducts ions in a desensitized state. Moreover, mutations Leu-255), where mutations reduce Ca2+ permeability and of two adjacent rings of leucines at the synaptic end of M2 (site affect other physiological properties of the response such as 2) have multiple effects. They abolish calcium permeability, its apparent affinity for ACh and the rate of currents onset increase the apparent affinity for acetylcholine by 10- to and desensitization. 100-fold, augment Hill numbers (up to 4.6-5.0) of acetylcho- line dose-response relationships, slow rates of ionic response onset, and lower the extent of desensitization. Mutations at MATERIALS AND METHODS these two topographically distinct sites within M2 selectively Mutagenesis. Mutants were prepared as described (23, 27). alter calcium transport without affecting the relative perme- Their coding sequence was checked. abilities for sodium and potassium. Electrophysiology. Oocytes were prepared, injected, and recorded as described (29). For voltage-clamp measure- Influx of calcium (Ca2+) through peripheral and brain nico- ments, cells were incubated in OR2 medium (solution B, tinic acetylcholine receptors (nAChRs) has been described in Table 1) and challenged by ACh application. Data from one several preparations including muscle (1, 2), chromaffin cells oocyte are given in the figures and values (mean ± SEM) (3, 4), cultured neurons from parasympathetic cardiac ganglia determined from 5 to 10 oocytes from more than one donor (5), rat medial habenular nucleus (6), and chicken ciliary are given in Table 2. ganglia (ref. 7; for review, see ref. 8). Neuronal nAChRs, in Current-voltage (I-V) curves were obtained by subtracting particular, exhibit a significant permeability to Ca2+ with passive membrane currents from currents evoked in the associated PCa/PNa close to 1 in parasympathetic cardiac presence of ACh. ACh was applied at a concentration close neurons (5) or 2.5 in PC12 cells (9), which contrasts with a to its EC50 value for each mutant. PCa/PNa of 0.1-0.3 in skeletal muscle cells (1, 2, 10). Computations were done by the constant field theory, Recent molecular cloning studies have led to the identifi- using the Goldman-Hodgkin-Katz equations for Na+, K+, cation of several genes encoding brain nAChR subunits that Ca2+, and Cl- permeabilities extended from that described in form functional acetylcholine (ACh)-gated ion channels upon ref. 30 for the N-methyl-D-aspartate receptor, to yield rela- expression in Xenopus oocytes (for review, see ref. 11). The tive permeabilities. Ionic activities were used in these equa- a3-f34 hetero- (12) and the a7 homo- (13) oligomers display, tions, instead of concentrations. Except for Mg2+, activity for instance, significant Ca2+ permeabilities (3, 14, 15). coefficients for Na+, Ca2+, and K+ were estimated as de- Chemical labeling (16-18) and site-directed mutagenesis scribed (30, 31) and are given in Table 1. Effects of all ionic (19-21) experiments have pointed to the critical role played substitutions were fully reversible. 1,2-Bis(2-aminophenoxy)- by the M2 segments in ion permeation through nAChR ethane-N,N,N' ,N'-tetraacetic acid (BAPTA) injections channels (for review, see ref. 22). In particular, they led to the were performed as described (14). identification of several rings of amino acids that contribute to the characteristic pharmacological (21, 23) and conduc- tance (20) properties of the ion channel, as its relative RESULTS selectivity among monovalent cations (24-26). In chicken Unless otherwise indicated, all experiments described below neuronal nicotinic a7 homooligomer, mutations ofthree rings were performed in the presence of internal BAPTA to sup- ofresidues within M2 (T244Q or L247T or V251T) resulted in press possible contribution of the Ca2+-activated chloride modifications ofthe response to ACh that were interpreted in currents as described (14, 15, 28, 31, 32). terms ofthe conversion of a high-affinity closed desensitized a7 Reversal Potential Is Sensitive to Calcium. When ex- state into a conducting state (23, 27, 28). In another study, the pressed in Xenopus oocytes, a7 wild-type (WT) receptors simultaneous introduction of three mutations in the M2 elicit rapidly desensitizing (Fig. 1A) inwardly rectifying ACh- segment of the a7 receptor converted its ionic selectivity evoked currents. To evaluate the relative permeability of a7 from cationic to anionic (14). For one of the mutations Abbreviations: ACh, acetylcholine; BAPTA, 1,2-bis(2-aminophe- The publication costs ofthis article were defrayed in part by page charge noxy)ethane-N,N,N',N'-tetraacetic acid; E,.,, reversal potential; payment. This article must therefore be hereby marked "advertisement" nAChR, nicotinic acetylcholine receptor; WT, wild type; I-V, cur- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rent-voltage; DHfSE, dihydro-f-erythroidine. 6971 Downloaded by guest on September 30, 2021 6972 Neurobiology: Bertrand et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 1. Solution composition (PCI/PNa = 0.05) nAChRs (5), was found sufficient to account Extracellular for the deviations detected at low K+ and Na+ concentra- concentration, mM Extracellular tions. Thus, with only Na+, K+, Ca2+, and Cl- as permeant ions, a full set of permeability ratios could be determined, Man activity, mM using the constant field theory (PNa/PK = 0.5, PCa/PK = 5, Solution Na K Ca nitol YNa YK YCa PC1/PK = 0.05; Table 2), that fitted the experimental data A (2x Na) 168.75 2.5 2.5 0.0 129.90 1.92 1.30 reported in Fig. 2 A and D-F. B (lx Na) 86.25 2.5 2.5 0.0 66.40 1.92 1.43 From these data, it appears that the a7 nAChR channel, C (0.02x Na) 1.75 2.5 2.5 82.5 1.35 1.92 2.05 like several other neuronal nAChRs (3, 5, 6, 9), is signifi- D (10x K) 1.75 25.0 2.5 82.5 1.35 19.25 2.05 cantly more permeable to Ca2+ than muscle nAChR (1, 2). Its E (3x K) 1.75 7.5 2.5 82.5 1.35 5.77 2.05 selectivity toward the monovalent cations Na+ and K+, on F (lx K) 1.75 2.5 2.5 82.5 1.35 1.92 2.05 the other hand, is comparable to that of endplate (1, 33) or G (isoCaCl2) 1.75 2.5 85.0 0.0 1.35 1.92 42.50 Torpedo electric organ receptor channels (24). H (10x Ca) 1.75 2.5 25.0 82.5 1.35 1.92 15.50 Mutation of Site 1 at the Cytoplasmic End of M2 Selectively I (8x Ca) 1.75 2.5 20.0 82.5 1.35 1.92 13.08 Alters Ca2+ Permeability. Mutation E237A was introduced at J (3x Ca) 1.75 2.5 7.5 82.5 1.35 1.92 5.55 the N-terminal end of M2, at a site (site 1) referred to as the K (lx Ca) 1.75 2.5 2.5 82.5 1.35 1.92 2.05 "intermediate ring of negatively charged residues" (20). L (0.4x Ca) 1.75 2.5 1.0 82.5 1.35 1.92 0.88 E237A and WT yield functional nAChRs eliciting ACh- M (0.1x Ca) 1.75 2.5 0.25 82.5 1.35 1.92 0.25 activated ionic currents of comparable amplitude, time All solutions contain 0.5 AM atropine and 5 mM Hepes (pH 7.4); course, and ACh sensitivity (in the presence of internal solutions A-G also contain 1 mM MgCl2; solutions A and B also BAPTA; see ref. 14). contain 1 mM phosphate. Ionic responses recorded at 200 ,uM ACh reverse at -24 mV under control conditions (Fig. 2B) and shift toward more WT to various monovalent and divalent cations, we mea- positive potentials upon a 2-fold increase in external NaCl (2 sured reversal potentials, at 100 ,uM ACh, after changing the x Na+; Erev = -3 mV). In contrast to WT, (i) replacement ionic composition of the external medium. As in studies on of NaCl with CaCl2 displaced the reversal potential toward muscle nAChR (2), Na+, K+, Ca2+, Mg2+, and Cl- were EK (isoCaC12; Ercv = -56 mV) and (ii) increasing CaCl2 taken into account as potentially permeant ions.
Load more

Recommended publications
  • The Excitable Cell. Resting Membrane Potential and Action Potential (1&2)
    The excitable cell. Resting Membrane Potential and Action Potential (1&2) Dr Sergey Kasparov School of Medical Sciences, Room E9 THIS TOPIC IS ALSO COVERED IN TUTORIALS! Teaching home page: http://www.bristol.ac.uk/phys-pharm/media/teaching/ Why do we call these cells “excitable”? Real action potentials.avi The key point: Bioelectricity is generated by the ions moving across cellular membranes Concentration of selected solutes in intracellular fluid and extracellular fluid in millimols mM 160 140 120 100 Insi de the cell 80 60 In extracellular fluid 40 20 0 1 The Na+/K+ pump outside inside [[mMmM]] + + [[mMmM]] + + + + + + + Na 145 + + 15 + K 4 + + 140 ATPATP ••isis an active ion transporter (ATP‐dependent) ••isis responsible for creating and upholding Na+ and K+ concentration gradients ••isis electrogenic – pumps 3 Na+ versus 2 K+ ions Reminder: Movement of ions is affected by concentration gradient and the electrical field Chemical driving force _ _ + + _ + + Electrical driving force _ + + _ + + + _+ _ + _ + + + + _ + _ + _ + + _ Net flux + + + + _ + _ + + V REMINDER: Diffusion through Ion Channels A leak channel A gated channel Both leak and gated channels allow movement of molecules (mainly inorganic ions) down the electrochemical gradient. So, if the gradient reverses, the ions will flow in the opposite direction. 1. The channels are aqueous pores through the membrane. 2. The channels are usually quite selective, for example some only pass Na+, others K+, still others – Cl- 3. Gated channels may be opened or closed by various factors
    [Show full text]
  • Synaptic Conductance Models
    Synaptic Conductance Models BENG/BGGN 260 Neurodynamics University of California, San Diego Week 4 BENG/BGGN 260 Neurodynamics (UCSD) Synaptic Conductance Models Week 4 1 / 14 Reading Material C. Koch, Biophysics of Computation, Oxford Univ. Press, 1999, Ch. 1, pp. 14-23 and 85-115. B. Hille, Ion Channels of Excitable Membranes, Sinauer, 2001, Ch. 6, pp. 169-100. A. Destexhe, Z.F. Mainen and T.J. Sejnowski, \Synthesis of Models for Excitable Membranes, Synaptic Transmission and Neuromodulation Using a Common Kinetic Formalism", J. Comp. Neuroscience, vol. 1, pp. 195-230, 1994. P. Dayan and L. Abbott, Theoretical Neuroscience, MIT Press, 2001, Ch. 5.8, pp. 177-178. BENG/BGGN 260 Neurodynamics (UCSD) Synaptic Conductance Models Week 4 2 / 14 Synaptic Transmission neurotransmitter vesicle receptor axon dendrite action postsynaptic potential t t current tpre presynaptic synaptic postsynaptic terminal cleft terminal Presynaptic action potential triggers release of neurotransmitter (through Ca2+) Postsynaptic binding of neurotransmitter at receptor induces opening of a channel leading to a postsynaptic current I Excitatory (AMPA, NMDA, ...) t EPSC ! EPSP I Inhibitory (GABAA, ...) t IPSC ! IPSP tpre BENG/BGGN 260 Neurodynamics (UCSD) Synaptic Conductance Models Week 4 3 / 14 Synapses are Stochastic Fig. 4.4 Postsynaptic Amplitude Is Highly Variable Fluctuations in the amplitude of EPSPs observed in a pyramidal cell by evoking an action potential in a nearby pyramidal cell. Both pairs of neurons are located in layer 2/3 of brain slices taken from rat visual cortex. (A) Four individual sweeps from the same synaptic connection. The EPSP amplitude over the entire population of cell pairs is 0:55 ± 0:49 mV.
    [Show full text]
  • Endogenous Serotonin Excites Striatal Cholinergic Interneurons Via The
    Neuropsychopharmacology (2007) 32, 1840–1854 & 2007 Nature Publishing Group All rights reserved 0893-133X/07 $30.00 www.neuropsychopharmacology.org Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the Activation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for Extrapyramidal Side Effects of Serotonin Reuptake Inhibitors 1 1,2 3 1 3 2 Paola Bonsi , Dario Cuomo , Jun Ding , Giuseppe Sciamanna , Sasha Ulrich , Anne Tscherter , 1,2 3 ,1,2 Giorgio Bernardi , D James Surmeier and Antonio Pisani* 1 2 Fondazione Santa Lucia I.R.C.C.S., European Brain Research Institute, Rome, Italy; Clinica Neurologica, Dipartimento di Neuroscienze, 3 Universita` di Roma ‘Tor Vergata’, Rome, Italy; Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath- applied serotonin (5-HT, 3–300 mM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, + + whose reversal potential was close to the K equilibrium potential. Accordingly, the involvement of K channels was confirmed either by + + increasing extracellular K concentration and by blockade of K channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons.
    [Show full text]
  • The Current-/Voltage-Clamp
    SimNeuron Tutorial 1 SimNeuron Current-and Voltage-Clamp Experiments in Virtual Laboratories Tutorial Contents: 1. Lab Design 2. Basic CURRENT-CLAMP Experiments 2.1 Action Potential, Conductances and Currents. 2.2 Passive Responses and Local Potentials 2.3 The Effects of Stimulus Amplitude and Duration. 2.4 Induction of Action Potential Sequences 2.5 How to get a New Neuron? 3. Basic VOLTAGE-CLAMP Experiments 3.1 Capacitive and Voltage-dependent Currents 3.2 RC-Compensation 3.3 Inward and outward currents. 3.4 Changed command potentials. 3.5 I-V-Curves and “Reversal Potential” 3.6 Reversal Potentials 3.7 Tail Currents 4. The Neuron Editor 5. Pre-Settings 2 SimNeuron Tutorial 1. Lab Design We have kept the screen design as simple as possible for easy accessible voltage- and current-clamp experiments, not so much focusing on technical details of experimentation but on a thorough understanding of neuronal excitability in comparison of current-clamp and voltage-clamp recordings. The Current-Clamp lab and Voltage-Clamp lab are organized in the same way. On the left hand side, there is a selection box of the already available neurons with several control buttons for your recordings, including a schematic drawing the experimental set-up. The main part of the screen (on the right) is reserved for the diagrams where stimuli can be set and recordings will be displayed. Initially, you will be in the Current-Clamp mode where you will only see a current-pulse (in red) in the lower diagram while the upper diagram should be empty. Entering the Voltage-Clamp mode you will find a voltage-pulse in the upper diagram (in blue) with an empty lower diagram.
    [Show full text]
  • Lecture 6 – Synaptic Transmission I -- Siegelbaum Postsynaptic
    Lecture 6 – Synaptic Transmission I -- Siegelbaum Postsynaptic Mechanisms 1. Stretch receptor reflex as a model of neuronal signaling How does an impulse get transmitted from one cell to the next? 2. Two major forms of synaptic transmission a. Electrical – fast, bidirectional (usually), no amplification. Mediated by gap junctions. b. Chemical – slower, unidirectional, amplification. mediated by presynaptic terminals & postsynaptic receptors. 3. Gap junctions – consist of pairs of channels that span pre and postsynaptic membranes. protein subunits called connexin. Six subunits make one hemi-channel, called a connexon. 4. Chemical synapses – two major types of postsynaptic receptors a. Ionotropic receptors – receptor and ion channel in same macromolecule. Transmitter binding opens a channel. b. Metabotropic receptors – coupled to G proteins and second messengers. Indirectly influence ion channels. 5. Neuromuscular junction – Action potential in presynaptic terminals causes calcium influx via voltage- gated calcium channels Æ release of transmitter acetylcholine (ACh) Æ ACh diffusion across synaptic cleft to postsynaptic membrane Æ ACh binds to and opens nicotinic ACh receptor (nAChR), a ligand-gated ion channel Æ leads to Na+ influx into muscle cell Æ large, suprathreshold fast excitatory post-synaptic potential (EPSP) in postsynaptic muscle at end-plate. Reduce size of EPSP with curare so that it is subthreshold. See passive decay of EPSP amplitude away from endplate. 6. Nicotinic ACh receptor – a pentamer composed of four types of homologous subunits (two α subunits, and one β,γ and δ subunit). Each subunit contains a large external domain, four transmembrane domains, termed m1-m4, and a short external C terminus. Two molecules of ACh bind to the receptor.
    [Show full text]
  • Serotonin and Cyclic Adenosine 3':Vmonophosphate Modulate The
    0270-6474/85/0507-1862$02.00/0 The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 5, No. 7, pi. 1862-1871 Printed in U.S.A. July 1985 Serotonin and Cyclic Adenosine 3’:VMonophosphate Modulate the Potassium Current in Tail Sensory Neurons in the Pleural Ganglion of ApJVsia ’ JONATHAN D. POLLOCK,’ LISE BERNIER,3 AND JOSEPH S. CAMARDO Department of Physiology, Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University and New York State Psychiatric Institute, New York, New York 10032 Abstract In sensory neurons of the abdominal ganglion of Aplysia califor- nica, which mediate the siphon-gill withdrawal reflex, serotonin elicits Tail sensory neurons in the pleural ganglion that mediate a slow, decreased conductance postsynaptic potential (slow PSP) the afferent portion of the tail withdrawal reflex in Ap/ysia by decreasing a specific potassium current (Klein et al., 1982; californica undergo heterosynaptic facilitation of transmitter Siegelbaum et al., 1982). This potassium current, called the S current, release during sensitization. As in the siphon sensory neu- is distinct from the fast potassium current (Connor and Stevens, rons, the transmitter serotonin produces facilitation and also 1971; Neher, 1971) the calcium-activated potassium current (Meech elicits a slow, decreased conductance excitatory postsyn- and Standen, 1975) and the delayed potassium current (Hodgkin aptic potential (EPSP) in these neurons. Using voltage clamp and Huxley, 1952; Thompson, 1977). The current is non-inactivating, and biochemical analyses, we have found that the slow EPSP and it participates in the resting and action potentials of the sensory in the pleural sensory neurons is due to a decrease in a neurons.
    [Show full text]
  • BI 360: Neurobiology Fall 2015
    BI 360: Neurobiology Fall 2015 Problem Sets: Questions and Answers These problems are provided to aid in your understanding of basic neurobiological concepts and to guide your focus for in-depth study. These practice questions are optional and answers will not be graded, although they will be discussed during discussion sections. The topics in these problem sets parallel those covered in lecture. Neurobiological concepts are complex and difficult, and oftentimes are not easily digested at first reading. We suggest that you review the course material several times by reading different texts/papers and talking through your difficulties with us and your fellow classmates. Stick with it B this material can be fully comprehended through persistent and focused effort. Problems listed below are in order of topics covered in lecture. Problem set numbers do not always correspond to specific lectures or weeks of the term. There are 5 problem sets in this series Problem Set 1 (IONS, MEMBRANES & EQUILIBRIA): 1.1 The equilibrium potential for sodium (ENa) = +58 mV if the concentration of Na on the outside of the cell is 10 times the concentration of Na on the inside of the cell. What happens to ENa if the extracellular concentration of sodium ([Na]o) is increased by a factor of 10? factor of 100? decreased by a factor of 10? Explain why ENa changes in the above examples in terms of the balance between chemical and electrical forces across the cell=s plasma membrane. Answer: The Nernst equation is the formula used to calculate the “equilibrium” or “reversal” potential for a particular ion given the relative internal and external concentrations of the ion.
    [Show full text]
  • Negative Shift in the Glycine Reversal Potential Mediated by Aca2ϩ- and Ph-Dependent Mechanism in Interneurons
    The Journal of Neuroscience, September 16, 2009 • 29(37):11495–11510 • 11495 Cellular/Molecular Negative Shift in the Glycine Reversal Potential Mediated by aCa2ϩ- and pH-Dependent Mechanism in Interneurons Yuil Kim1 and Laurence O. Trussell2 1Neuroscience Graduate Program, Oregon Health & Science University, and 2Oregon Hearing Research Center/Vollum Institute, Portland, Oregon 97239 Cartwheel cells are glycinergic auditory interneurons which fire Na ϩ- and Ca 2ϩ-dependent spike bursts, termed complex spikes, and which synapse on both principal cells and one another. The reversal potential for glycine (Egly ) can be hyperpolarizing or depolarizing in cartwheel cells, and many cells are even excited by glycine. We explored the role of spike activity in determining Egly in mouse cartwheel cells using gramicidin perforated-patch recording. Egly was found to shift toward more negative potentials after a period of complex spiking or Ca 2ϩ spiking induced by depolarization, thus enhancing glycine’s inhibitory effect for ϳ30 s following cessation of spiking. 2ϩ Combined perforated patch electrophysiology and imaging studies showed that the negative Egly shift was triggered by a Ca -dependent Ϫ Ϫ intracellular acidification. The effect on Egly was likely caused by bicarbonate-Cl exchanger-mediated reduction in intracellular Cl ,as Ϫ Ϫ H2DIDS and removal of HCO3 /CO2 inhibited the negative Egly shift. The outward Cl flux underlying the negative shift in Egly opposed a positive shift triggered by passive Cl Ϫ redistribution during the depolarization. Thus, a Ca 2ϩ-dependent mechanism serves to maintain or enhance the strength of inhibition in the face of increased excitatory activity. (Farrant and Kaila, 2007).
    [Show full text]
  • The Properties and Regulation of Functional Acetylcholine Receptors on Chick Ciliary Ganglion Neurons
    The Journal of Neuroscience, November 1967, 7(11): 3612-3622 The Properties and Regulation of Functional Acetylcholine Receptors on Chick Ciliary Ganglion Neurons Joseph F. Margiotta, Darwin K. Berg, and Vincent E. Dionne Departments of Pharmacology and Biology, University of California, San Diego, La Jolla, California 92093 The properties of acetylcholine receptor (AChR) channels are also found in many areas of the nervous system, where they on chick ciliary ganglion neurons in culture were examined mediate synaptic transmission between neurons. Although ini- using patch-clamp recording techniques. Acetylcholine (ACh) tial studies indicated that neuronal AChRs share some of the was applied by rapid microperfusion. Whole-cell current noise basic functional properties of their counterparts on skeletal mus- analysis revealed a single class of functional receptors on cle fibers (Rang, 198 1; Derkach et al., 1983; Ogden et al., 1984) the neurons. Dose-response studies indicated a & of about significant immunological, pharmacological, and structural dif- 36 PM and a Hill coefficient of 1.5-1.7, predicting 2 ACh ferences have also been reported (Patrick and Stallcup, 1977a, binding sites per receptor. Both fast and slow components b; Carbonetto et al., 1978; Ascher et al., 1979; Boulter et al., of receptor desensitization were observed. Single-channel 1986). Because of these differences, rules that govern the reg- recordings from excised outside-out patches of soma mem- ulation of AChR function, number, and distribution in muscle brane exposed to 2-5 PM ACh indicated a single-channel may not apply to neurons. Information about neuronal AChR conductance of 40 pS, a reversal potential of -9 mV, a mean regulation has been limited both because newly developed re- open duration of 1 msec, and an opening probability of 0.34.
    [Show full text]
  • Synapses Models of the Synapse
    Department of Mathematical Sciences B12412: Computational Neuroscience and Neuroinformatics Synapses Models of the synapse The contacts of the axon to target neurons are either located on the dendritic tree or directly on the soma, and are known as synapses. Most synapses are chemical contacts, that is, the arrival of an action potential at the synapse induces the secretion of a neurotransmitter that in turn leads to a change in the potential of the membrane of the target neuron. Depending on the type of synapse, an incoming pulse either causes an increase in electrical potential (excitatory synapse) or a decrease (inhibitory synapse). Left: Schematic diagram showing release of neurotransmitter as vesicle membrane fuses with end bulb membrane. Right: Electron micrograph showing how synaptic vesicles fuse with cell membrane as neurotransmiter is discharged. At a synapse presynaptic firing results in the release of neurotransmitters that causes a change in the membrane conductance of the postsynaptic neuron. This postsynaptic current may be written mathematically as Is = gss(Vs - V) where V is the voltage of the postsynaptic neuron, Vs is the membrane reversal potential and gs is a constant. The term Vs - V is often called a shunt as it acts to push the voltage toward the synaptic reversal potential Vs. The variable s corresponds to the probability that a synaptic receptor channel is in an open conducting state. This probability depends on the presence and concentration of neurotransmitter released by the presynaptic neuron. The sign of Vs relative to the resting potential (assumed to be zero) determines whether the synapse is excitatory (Vs > 0) or inhibitory (Vs < 0).
    [Show full text]
  • Neuroscience 201A Exam “Key”, October 7, 2014
    Name: _____KEY_________________________ Neuroscience 201A Exam “Key”, October 7, 2014 Question #1 7.5 pts Consider a spherical neuron with a diameter of 20 µm and a resting potential of -70 mV. If the net negativity on the inside of the cell (all of the “excess” anions) were uniformly distributed within the volume of the cell rather than being “glommed” onto the inner surface of the plasma membrane, what would the concentration of these ions be (in M)? Assume that these are all univalent ions. Assume that the capacitance of the plasma membrane is the standard 1 µF/cm2. Page 1 Name: _____KEY_________________________ Question #2 12.5 pts Imagine that you have a squid neuron with a single population of leak channels, which are cation channels with permeability to only Na+ and K+. The concentrations of permeant ions on each side of the membrane are as follows: Ion Na+ K+ Outside 550 mM 20 mM Inside 55 mM 440 mM a) If the resting membrane potential of the cell is -63 mV, calculate gNa/gK for the leak channels. (You are encouraged to use conductances for this problem (not permeabilities). b) If the squid neuron is spherical with a diameter of 200 µm, if the leak conductance of its membrane is 0.3 mS/cm2, and if the conductance of a single leak channel is 10 pS, calculate the average density of leak channels in the membrane, as # per µm2. Page 2 Name: _____KEY_________________________ Question #3 12.5 pts The above figure illustrates a set of current responses (a, lower traces) to voltage steps (a, top traces) for a population of channels in a neuron with physiological solutions bathing the inside and outside of the cell.
    [Show full text]
  • GABA Reversal Potential Is Different in Perforated Vs. Whole-Cell Patch Recording Methods
    GABA reversal potential is different in Perforated vs. Whole-cell patch recording methods Gramicidin forms membrane pores that are exclusively permeable to monovalent cations and small, uncharged molecules, thus leaving internal Cl- concentration undisturbed (E GABAA Equilibrium Potential GABAA) is determined by two methods at various ages. Responses shift from monophasic to a more complicated trace due to maturation of GABAB receptor subtype GABAA equilibrium potential declines over development, perhaps due to a fall in chloride ion concentration inside the cell. The chloride ion is the principle ion mediating GABAA receptor effects: the chloride gradient contributes significantly to E GABAA. Whole-cell recordings used to Perforated patch recordings No developmental shift in EGABA bias the internal chloride conc. A Furosemide (chloride to that of the pipette, and muscimol using whole-cell recording. transporter blocker) reduces (agonist) induced reversal potentials GABA equilibrium potential. were measured. These values are A compared to the reversal potential for chloride predicted by Nernst eq. More evidence for GABAA mediated membrane depolarization in early development : GABAA agonist causes reversible calcium entry through voltage-gated Ca++ channels. The reversal potential for endogenously active GABAA-mediated synaptic current (sPSC) is nearly identical to the reversal potential for the exogenously applied GABAA agonist Muscimol. These are not mini’s: TTX abolishes them These sPSC’s are not glutamatergic CNQX and APV do not block. BMI= Bicuculline methiodide, GABAA receptor antagonist. A. whole-cell current clamp B. Perforated patch voltage clamp. Averaged sPSC’s Muscimol induced Spontaneous increases in intracellular calcium (measured by confocal calcium imaging) are mediated by GABAA receptor activation.
    [Show full text]