Molecular Neurophysiology PDF

Molecular Neurophysiology: From Molecules to Systems Jochen Winterer Lab of Systems Neuroscience / Institute for Neuroscience / ETH email: [email protected] ...

Molecular Neurophysiology: From Molecules to Systems Jochen Winterer Lab of Systems Neuroscience / Institute for Neuroscience / ETH email: [email protected]  Synaptic Complex in the last lecture we have learned how an electrical signal, the presynaptic action potential, is coded into a Presynaptic element chemical signal (neurotransmitter release). in this lecture we will deal with the postsynaptic side of Synaptic cleft our synaptic complex and how the chemical signal is coded back into an electrical signal. Postsynaptic element Adapted from Korte, M., Schmitz, D., 2016. Physiol Rev 96  Postsynaptic and Transsynaptic Signaling Molecular Neurophysiology: From Molecules to Systems Jochen Winterer Lab of Systems Neuroscience / Institute for Neuroscience / ETH Canonical design of a central synapse Postsynaptic receptors Postsynaptic scaffold (or postsynaptic density) Synaptic cell adhesion molecules Sudhof, 2018, Neuron  Postsynaptic receptors of the amino acid neurotransmitters Glutamate and GABA Glutamate and GABA are by far the most abundant excitatory and inhibitory neurotransmitter in mammalian brain, resp. Glutamate receptors GABA receptors ionotropic receptors ionotropic receptors metabotropic receptors metabotropic receptors ligand-gated ion channels ligand-gated ion channels AMPA-Rs mGlu GABAA-Rs GABAB -Rs Rs NMDA-Rs Kainate-Rs Glutamatergic and GABAergic synaptic transmission GABA depolarizing -50mV -50mV -60mV -60mV -70mV hyperpolarizing -70mV Hammond, Cell. and Mol. Neurophysiology, 2024  Synaptic transmission Synapses are fundamental computational units that wire neuronal circuits. Diverse computational properties of synapses are shaped by the precise interactions between pre- and postsynaptic neurons. Computational diversity is vastly achieved via different neurotransmitters and their corresponding receptors (excitation/inhibition). Biophysical properties of a receptor dictate the consequences of receptor activation: which ion fluxes are responsible for depolarizing or hyperpolarizing the postsynaptic membrane? How to depolarize / hyperpolarize a cell? depolarization -50mV This is not obvious: -60mV -70mV depolarization [hyperpolarization] of the neuron could be due to efflux [influx] of GABA negatively charged ions (anions) or influx [efflux] of positively charged ions (cations)! -50mV -60mV hyperpolarization -70mV Hammond, Cell. and Mol. Neurophysiology, 2024  Questions / Learning objectives What are the properties of the postsynaptic receptors to allow for depolarization/hyperpolarization of the postsynaptic membrane? How can we characterize the properties of these receptors – how to measure? How are those receptors anchored/stabilized in the postsynaptic membrane? What is the role of synaptic cell adhesion molecules, and what is their impact on synaptic transmission?  How to measure AMPA-R mediated excitatory postsynaptic potentials/currents Whole Cell patch-clamp Patch Clamp amplifiers give the possibility to record: the voltage of the cell membrane in Current- clamp mode (1 and 2). -60mV the current that is flowing over the membrane in Voltage-clamp mode (3). Extracellular solution +10mV -60mV In Current-clamp mode we «follow» the membrane potential: voltage follower. 3 0pA -200pA In Voltage-clamp mode we record the current that the amplifier needs to apply to keep the voltage at a given potential. Hammond, Cell. and Mol. Neurophysiology, 2024  Characterizing the properties of AMPA-R: or to what are they permeable? Receptor activation by quisqualate Recording Vh= 60mV Vh= - 60mV How can we interpret these results? Hammond, Cell. and Mol. Neurophysiology, 2024  I/V curve Outward current iq (pA) Current / Voltage (I/V) curve Relationship between the current and voltage Voltageholding Vh (mV) Describes the current flow at a specific voltage Be aware: outward current is by convention a positive current value! It can arise from the movement of positive ions Inward current iq (pA) out or negative ions into the cell! (voltage clamp configuration!). Inward current is therefore a negative current value. Hammond, Cell. and Mol. Neurophysiology, 2024  Quick recap: ion concentrations and membrane potential Contribution of the concentration gradient Contribution of the electrical gradient Hammond, Cell. and Mol. Neurophysiology, 2024 Quick recap: ion concentrations and membrane potential when membrane potential (Vm) is different from equilibrium potential (Eion) there is a passive diffusion of this ion through an open channel. The difference (Vm - Eion) is called the electrochemical gradient. Equilibrium potential (Eion) or reversal potential: net ion flux is null. with the voltage clamp method we can clamp the membrane potential (Vh) and measure the current flow at (Vh). by changing the concentration of ions [X]i or [X]e we will change Eion of that particular ion. Hammond, Cell. and Mol. Neurophysiology, 2024  Characterization of the AMPA-R Outward Iq (pA) 1.0 0.5 We obtain unitary currents Iq (pA) as a function of the holding membrane potential VH (mV). -80 -40 40 80 Vm (mV) -0.5 The current Iq reverses at a value close to 0mV: “reversal potential” or -1.0 equilibrium potential (Eion). Inward Iq (pA) 𝑹𝑻 Nernst Equation: Eion= 𝒛𝑭 𝐥𝐧( 𝒊𝒐𝒏 𝒆/ 𝒊𝒐𝒏 i) Intra (in mM): Extra (in mM): [K+]i=140 [K+]e=3 [Na+]i=14 [Na+]e=140 [Cl-]i=14 [Cl-]e=146 [Ca2+]i=0.0001 [Ca2+]e=1.5 This suggests that the quisqualate-activated channel is permeable to K+ and Na+ !  Characterization of the AMPA-R Outward Iq (pA) 1.0 0.5 If we reduce the extracellular Na+ concentrations from 140mM to 50mM we get a shift in the reversal potential (from 0 to -20mV). -80 -40 40 80 Vm (mV) -0.5 These results suggest that the quisqualate-activated channel is -1.0 permeable to Na+, K+ ions (monovalent ions). Inward Iq (pA)  Summary The native quisqualate-activated channel is permeable to monovalent cations, we now know that the neurotransmitter is glutamate and that the channel is an AMPA receptor. The application of quisqualate at a membrane potential of Vm = -60mV evokes a unitary inward current that results from the inflow of Na+ ions and an outflow of K+ ions through the same channel (the Na+ inflow is stronger than the K+ outflow). This AMPA receptor is a classic cationic channel receptor: It has a negligible permeability to Ca2+ ions.  Structure and subunit composition of AMPA-R Amino-terminal domain AMPA-receptors are tetramers. Four subunits: GluA1, GluA2, GluA3 Ligand-binding and GluA4. domain Subunits are differentially expressed ions (most common is GluA1 containing). Transmembrane domain AMPA-R can be devided into two functionally Carboxy-terminal distinct assemblies based on the inclusion or domain exclusion of the GluA2 subunit. Hammond, Cell. and Mol. Neurophysiology, 2024  AMPA-R editing GluA2 is edited at the M2 transmembrane domain. Editing is a posttranscripional change in the premRNA sequence: (DNA sequence is different from the mRNA sequence). The codon 586 glutamine (Q) is changed to arginine (R) (Q/R site). GluA2 lacking AMPA receptors are Ca2+ permeable, whereas GluA2 containing AMPA receptors are Ca2+ impermeable. Hammond, Cell. and Mol. Neurophysiology, 2024 Channel Conductance of GluA2 containing vs. GluA2 lacking AMPA receptors GluA2 lacking AMPA receptors have an increased channel conductance as compared to GluA2 containing AMPA receptors GluA2-editing in amyotrophic lateral sclerosis (ALS) ALS is a fatal motor neuron disease. It causes progressive degeneration of motor neurons in the spinal cord and brain. Reduced expression of the RNA editing enzyme ADAR2 in sporadic ALS, responsible for adenosine-to-inosine conversion at the GluA2 glutamine/arginine (Q/R) site. Consequently, AMPA-R that express the unedited form of GluA2 and are permeable to Ca2+. Ca2+ is an important signaling molecule and second messenger. However, too much Ca2+ is toxic for neurons and Ca2+ excitotoxicity represents a likely candidate of cell death observed in ALS.  Summary AMPARs are grouped into two functionally distinct tetrameric assemblies based on the inclusion or exclusion of the GluA2 receptor subunit. GluA2-containing receptors are thought to be the most abundant AMPARs in the CNS, typified by their small unitary events and Ca2+ impermeability. In contrast, GluA2-lacking AMPARs exhibit large unitary conductance and marked Ca2+ permeability. In sporadic ALS AMPA-R express the unedited form of GluA2 and are therefore permeable to Ca2+. Ca2+ excitotoxicity represents a likely candidate of cell death of motorneurons observed in ALS.  GABAA-R mediated inhibitory postsynaptic potential/current i(pA) 3 2 1 Vm (mV) -100 -50 50 100 -1 -2 -60mV -40 -3 -60mV -60 -10mV -100 Eion is at -60mV 1pA +200pA 3 0pA Hammond, Cell. and Mol. Neurophysiology, 2024  Characterizing GABAA-R mediated ion flux 3 i(pA) 2 1 146 mM -100 -50 50 100 -1 Vm (mV) 14 mM -2 -3 𝑹𝑻 Nernst Equation: Eion= 𝐥𝐧( 𝒊𝒐𝒏 𝒆/ 𝒊𝒐𝒏 i) 𝒛𝑭 −𝟎. 𝟎𝟔 = ± 𝟎. 𝟎𝟑 𝐥𝐧( 𝒊𝒐𝒏 𝒆/ 𝒊𝒐𝒏 i) The GABAA receptor is permeable to Cl- −𝟎. 𝟎𝟔 = −𝟎. 𝟎𝟑 𝐥𝐧(𝟏𝟎) Hammond, Cell. and Mol. Neurophysiology, 2024 Intracellular Cl- concentration dictates polarity of the current through GABAA receptors 3 3 i(pA) i(pA) 2 2 1 Vm (mV) 1 Vm (mV) -100 -50 50 100 -100 -50 50 100 -1 -1 146 mM -2 146 mM -2 -3 -3 14 mM 146 mM GABA can be depolarizing! 0mV -50mV -50mV -60mV Hammond, Cell. and Mol. Neurophysiology, 2024  GABA is depolarizing during development mature immature I/V for AMPA receptor Is the GABAA channel permeable to cations in immature neurons ? Or is the GABAA channel permeable to Cl- in immature neurons, but the Cl- driving force is reversed due to an increased [Cl-]intra ? Hammond, Cell. and Mol. Neurophysiology, 2024 EGABA during development Increase in EGABA results from an increase in intracellular Cl-: shift in EGABA early expression of the chloride importer NKCC1 in immature neurons, while the main chloride exporter KCC2 has a delayed expression. In the former case, GABA will even generate APs, and in the latter, it will inhibit their generation. Hammond, Cell. and Mol. Neurophysiology, 2024  Summary The GABAA receptor channel is activated by GABA, the main inhibitory neurotransmitter in the mammalian central nervous system. Depending on the membrane potential and the concentration of Cl- in the extracellular and intracellular media, there is an influx or an efflux of Cl-. In adult neurons, there is generally an influx of Cl-, that is, an outward current which hyperpolarizes the membrane. This hyperpolarization is the IPSP mediated by GABAA receptors. In young neurons GABA can be depolarizing: there is a higher Cl- concentration intracellularly than in adult neurons due to delayed expression of the main chloride exporter KCC2. Synapse-driven events are generated with GABAergic interneurons developing before principal glutamatergic pyramidal neurons.  The NMDA-Receptor the NMDA-R mediated EPSP is much broader! Hammond, Cell. and Mol. Neurophysiology, 2024 Characterization of the NMDA-Receptor [0 Mg2+]out !!! Hammond, Cell. and Mol. Neurophysiology, 2024  Effect of physiological [Mg2+]out on the NMDA-R NMDA channel is subject to a voltage-dependent block by Mg2+. Hammond, Cell. and Mol. Neurophysiology, 2024 The NMDA channel is highly permeable to monovalent cations and to Ca2+ Hammond, Cell. and Mol. Neurophysiology, 2024  Summary The NMDA receptor channels are unique among the glutamate receptors since their ion channel is subject to a voltage-dependent block by Mg2+. they are highly Ca2+ -permeable. they display unusually slow kinetics owing to slow glutamate unbinding. they play a pivotal role in long-term synaptic plasticity (lecture: Synaptic plasticity 1: Facilitation, LTP, LTD by Roberto Fiore) Definition of anatomical and functional parts of a synapse Byczkowicz et al., 2018, Neuroscience Research 127 Postsynaptic scaffold Postsynaptic scaffold (or density; PSD) is a specialization of the postsynaptic membrane. The PSD is located at the tip of the dendritic spine, opposing the presynaptic terminal. Membrane-Associated Guanylate Kinase proteins, MAGUKs (e.g. PSD95), are interacting with their PDZ domains with iGLURs. PDZ domains are protein-protein interaction modules that recognize short amino acid motifs at the C- termini of target proteins. Bessa-Neto and Choquet. 2023, Molecular and Cellular Neuroscience MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density Chen at al., 2015, PNAS Transsynaptic Signaling Synaptic cell adhesion molecules (synaptic CAMs, or simply SAMs) are synaptic «organizers». They are thought drive synapse maturation and control the properties of synapses. SAMs are signaling in both directions, pre- and postsynaptically Sudhof, 2017, Cell SAMs, the “making” and “shaping” of synapses ← shaping → ← making → Sudhof, 2021, Journal of Cell Biology  Cell-adhesion molecules and the postsynaptic density protein Shank in neurodevelopmental disorders Autism Spectrum Disorder (ASD): NLGNs, NRXNs, SHANKs. Schizophrenia: NRXNs. Epilepsy: NRXNs, SHANKs Intellectual Disability: NLGNs, NRXNs, SHANKs. Sudhof, 2017, Cell  Summary PSD is a morphological and functional specialization of the postsynaptic membrane. PSD is essential for anchoring of postsynaptic neurotransmitter receptors. Synaptic cell adhesion molecules (SAMs) provide transsynaptic signaling SAMs can build up synapses and they can modulate synaptic properties Many SAMs and postsynaptic density proteins are implicated in neuropsychatric disorders Thanks, we are done !