Na /Cl - -dependent Neurotransmitter Transporters Stine Meinild Lundby The Neurobiology Group Molecular, Cellular, and Integrative Physiology Section November 26 th, 2007 slide 1 Outline Synaptic neurotransmission Family of Na /Cl - dependent neurotransmittor transporters Function of Na /Cl - dependent neurotransmittor transporters Monoamine transporters transporters Known and development of new drugs Research on Na /Cl - dependent neurotransmittor transporters November 26 th, 2007 Slide 2 Synaptic Transmission http://outreach.mcb.harvard.edu/animations/synaptic.swf Excitatory actions at synapses result from opening of channels permeable to both Na and K Inhibitory actions at synapses result from opening of channels permeable to Cl - the nature of the neurotransmitter has no influence on the kind of synaptic signal How did the neurotransmitter cross the membrane? November 26 th, 2007 Slide 3 1
transporters November 26 th, 2007 Slide 4 The family of Na /Cl - -dependent neurotransmitter transporters Includes the transporters for: - dopamine - serotonin Excitatory - norepinephrine - - betaine Inhibitory - glycine and also proline, taurine, creatine, cationic amino acids (B[0]) and orphans Homology to transporters with unknown function in a broad range of bacteria (at least 40-50 different) Targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. November 26 th, 2007 Slide 5 Na /Cl - -dependent neurotransmitter transporters - approximately 650 amino acids - 12 transmembrane segments - high homology - coupling of downhill transport of Na to uphill transport of neurotransmitter - cotransport of Na - dependence of Cl - November 26 th, 2007 Slide 6 2
Monoamine Transporters Dopamine Norepinephrine Serotonin NH 2 NH 2 CH 2CH 2NH 2 N H The Dopamine Transporter (DAT) The Norepinephrine Transporter (NET) The Serotonin Transporter (SERT) - only three monoamine transporters have been identified! November 26 th, 2007 Slide 7 Monoamine Transporters Dopaminergic neurons VTA Substantia Nigra NE ergic neurones Locus ceruleus Serotonergic neurones Raphe Nucleus The Dopamine Transporter (DAT) The Norepinephrine Transporter (NET) The Serotonin Transporter (SERT) November 26 th, 2007 Slide 8 Monoamine Transporters Cocaine Amphetamine Cocaine Amphetamine Tricyclic antidepressants (imipramine,desipramine) Cocaine Amphetamine SSRI s (fluoxetin, sertraline, citalopram, paroxetin) The Dopamine Transporter (DAT) The Norepinephrine Transporter (NET) The Serotonin Transporter (SERT) November 26 th, 2007 Slide 9 3
The Serotonin Transporter is target of the SSRI s Depression Anxiety Post Traumatic Syndrome Obsessive Compulsive Disorder Bulimia Nervosa Selective Serotonin Reuptake Inhibitors SSRI s Prozac Citalopram November 26 th, 2007 Slide 10 http://www.prozac.com/how_prozac/how_it_works.jsp?reqnavid=2.2 Dopaminergic neurotransmission Physiologic processes: reward addiction movement lactation Improper dopaminergic tone Schizophrenia Parkinson's disease drug addiction Parkinson Loss of dopaminergic cells in Substantia Nigra loss of motor function Schizophrenia Malfunction of dopaminergic cells in VTA (ventral tegmentalt area) 1) hallucinations 2) impairred cognition November 26 th, 2007 Slide 11 DAT (dopamine transporter) knock-out mice Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter November 26 th, 2007 Giros et al., Nature 1996 Slide 12 4
Dopamine Transporter - Identification of substrate binding sites Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. November 26 th, 2007 Loland, C. J. et al. J. Biol. Chem. 2004;279:3228-3238 Slide 13 ergic neurotranmission November 26 th, 2007 Slide 14 Transporters Betaine β-alanine β-alanine O NH 2 O N O NH 2 O NH 2 GAT-1 GAT-2 GAT-3 GAT-4 (BGT-1) November 26 th, 2007 Slide 15 5
Transporters characteristic brain regions Cerebral cortex Cerebellar cortex Basal forbrain Cerebral cortex Cerebellum Basal stem Leptomenigeal cells Thalamus Choroid plexus Hypothalamus Amyddala Choroid plexus, corpus callosum GAT-1 GAT-2 GAT-3 GAT-4 (BGT-1) November 26 th, 2007 Slide 16 Transporters inhibitors November 26 th, 2007 Slide 17 ergic neurotransmission Generally, increased transmission will depress neuronal activity. Accordingly, drugs that increase inhibitory activity are used as anxiolytics, hypnotics, anesthetics, anti-epileptics and muscle relaxants. Epilepsy: November 26 th, 2007 Slide 18 6
What would we like to learn about the transporter Structural dynamics of the transport process Identify and characterize the substrate binding sites Characterize new sub-type specific inhibitors November 26 th, 2007 Slide 19 Xenopus Oocytes Expression System Two happy female Xenopus frogs about to help science. Harvest oocytes Inject crna coding for protein of interest crna is transcribed into protein and expressed in membrane 10 cm November 26 th, 2007 Slide 20 Electrophysiology using Xenopus oocytes Mount oocyte in recording chamber. Clamp membrane potential in real-time!!!!! Record transmembrane current in response to superfusate 50 na 40 s 100 mm Na 1 mm -100 na November 26 th, 2007 Slide 21 7
Wave Start 1 2 3 4 Wave End Recordings with Xenopus Oocytes Current trace Clamp membrane potential Detect current with time Change composition of perfusing solution Example: Oocyte injected with GAT1 crna ChoCl V hold -50 mv 100 na 100 sec November 26 th, 2007 Slide 22 Recordings with Xenopus Oocytes Voltage jumps; I/V curves steady state kinetics Jump V m to V test (50 to -150 mv; 20 mv increments) Detect current with time at each V test Plot steady state current as function of V test 200 Steady-state current Cmd 0 (mv) 0 -induced current V m (mv) -200 0 400 Time (ms) 800-150 -110-70 -30 10 50-100 -200-300 I (na) -400-500 November 26 th, 2007 Slide 23 Electrophysiological Characterization of hgat2 November 26 th, 2007 Slide 24 8
Substrate induced currents NaCl β-ala betaine V m (mv) 20-150 -110-70 -30 10 50 betaine -20 20 na 50 s β-alanine -40-60 -80-100 I (na) Christiansen et al. 2007 November 26 th, 2007 Slide 25 Substrate affinities and voltage dependence # 5 hgat2 β-alanine-induced current # 5 hgat2 β-alanine-activation V 50 m (mv) -100-50 50-50 -100-150 I (na) 0µM 1µM 3µM 10µM 30µM 100µM 300 µm 1000µM 3000µm 10000µM -I (na) 150 100 50 0 0 250 500 750 1000 [β-alanine] (µm) -110.0-90.0-70.0-50.0-30.0-10.0 10.0 30.0 50.0 K ½ β-alanine The Michaelis-Menten equation: I = (I max [S])/([S]K 0.5) 80 60 40 K ½ (µm) K½ (β-alanine) 20-110 -70-30 10 50 Vm (mv) November 26 th, 2007 Slide 26 Ion-dependence NaCl NaMES CholCl 100 80 I/I max (%) 60 40 20 10 na 50 s 0 NaCl - Low Cl No Na Christiansen et al. 2007 November 26 th, 2007 Slide 27 9
SNAP a potential inhibitor SNAP NaCl 50 µm 100 µm 500 µm I/I max (%) 100 80 60 40 10 na 20 s 20 0 bf SNAP Christiansen et al. 2007 November 26 th, 2007 Slide 28 November 26 th, 2007 Slide 29 Structural Dynamics of Membrane Transport Proteins November 26 th, 2007 Slide 30 10
Voltage Clamp Fluorometry Vcom _ Voltage-Clamp Fluorescence Microscope hν Dichroic Mirror Detector November 26 th, 2007 Slide 31 Summary Na /Cl - -dependent neurotransmitter transporters are important for neurotransmission Na /Cl - -dependent neurotransmitter transproters are important drug targets Na /Cl - -dependent neurotransmitter transproters interesting to study master projects available contact: smeinild@aki.ku.dk Stine: 3532 1651 lokale 336 November 26 th, 2007 Slide 32 11