Neuro Lecture #2 PDF

Lecture 011625 Bioelectric Circuits and Ion Channels – Electrophysiological Studies Reading: – Molecular Approach Ch 2, 3 – Types of Channels and t...

Lecture 011625 Bioelectric Circuits and Ion Channels – Electrophysiological Studies Reading: – Molecular Approach Ch 2, 3 – Types of Channels and their Gating Membrane potential – Electrochemical equilibrium – Resting membrane potential No one has the ‘capacity’ to ‘resist’ the lure of neuroscience! – Selective permeability – Driving Force – Nernst and Goldman equations Electrotonic (passive) properties – Time and length constants - first look Action potential introduced  Membrane Potentials A. Recording of Membrane Potential Δ VM Changes in VM from rest include: A. B. Graded response i.e. synaptic potential C. Active response i.e. action potential Resting Membrane Potential VM Inside relative to Outside At rest, cells have negative VM  Study of ion channels - electrophysiology Intracellular recording Patch clamp recording current clamp form of voltage clamp measures membrane potential VM measures ionic current Recordings and conventions (voltage) IK+ INa+ ICa++ = membrane potential across membrane: Recordings and conventions (current): depolarization VM + (“upwards” trace) inward + current (“downwards” trace) hyperpolarization VM - (“downwards” trace) outward + current (“upwards” trace) + - Membrane Potential = VM Ionic Current = I(ION)  Intracellular recording of resting and action potentials inject positive current - VM more positive = depolarization inject negative current - VM more negative = hyperpolarization threshold level of depolarization leads to action potential(s) What is the basis for RP? What is the basis for AP? What are their roles?  Noble laureates: Patch clamp recording of single voltage-gated Na+ channel Sakmann and Neher stimulus = membrane depolarization http://www.nobelprize.org/nobel_prizes /medicine/laureates/1991/sakmann.jpg effect = channel opening, Na+ influx http://www.nobelprize.org/nobel_prizes /medicine/laureates/1991/neher.jpg Ionic current is sum of flux through multiple channels voltage current How do voltage-gated channels work? What are their functions? inward current = flux of (+) IN (creates membrane depolarization)  Nerve cell membrane Lipid Bilayer Pumps Ion channels 1 1. LIPID BILAYER 2. PUMPS barrier to the diffusion of ions transport ions to create concentration gradients 2 3 3. CHANNELS embedded in phospholipid bilayer selective for K+, Na+, Ca++, or Cl- some are less selective – nicotinic AchR passive flux of ion - diffusion gated by varied stimuli change in VM, transmitters, mechanical stimuli, or intracellular biochemicals channels regulate resting and action potentials  1. Which ion is permeant? Selectivity Na+, Ca++ channels - positive influx of ion K+ channels - positive efflux of ion Cl- channels - negative influx of ion 2. What stimulus triggers opening? Gating Types of 2A. BIOCHEMICAL-GATED*** gated by G proteins, cyclic nucleotides, Ca2+ ion channels * focus Module I acting DIRECTLY to open channel *** focus Module III 2B. LIGAND GATED* (Graded potential change) Neurotransmitter binds to receptor /channel complex conformational change in receptor is coupled to channel opening or to second messenger system. Example: Ach receptors. 2C. VOLTAGE-GATED* (Action potential) Alteration in membrane potential moves voltage sensor conformational change in sensor is coupled to channel opening 2D. MECHANOSENSITIVE*** Mechanical stretch enlarges central region - channel opens 3. What are kinetics of channel? How long it takes to open How long it takes to inactivate How long it takes to recover from inactivation Intracellular recording of resting membrane potential = EM or VM cell is hyperpolarized at rest; values range from -40 mV to -100 mV (a depolarized cell is when VM becomes more positive) VM = Potential difference across the cell membrane - charge separation at membrane What does the reading on the oscilloscope (voltmeter) mean? Basis of Membrane Potential 1. Role of sodium / potassium pump 1. Distribution of ions [Na+] high OUT, [K+] high IN 2. Separation of charge 3 Na+ OUT, 2 K+ IN 3. ELECTROGENIC PUMP Direct effect is only -5 mV to -10 mV of -75 mV resting potential 4. Remainder (most) of EM (VM) ionic flux through channels Na+ inward: EM (VM) more positive K+ outward: EM (VM) more negative 2. Role of ion channels 1. Electrochemical equilibrium concentration gradient electrical gradient EION is equilibrium potential for ion 2. Selective permeability PION Nernst equation: EION = +58 mV log { IONout } IONin Possible contribution of ion to EM IF that ion is permeant Goldman-Hodgkin-Katz equation: EM =  {PION x EION} Membrane potential EM is the sum of all equilibrium potentials EION as function of their permeability PION Resting membrane potential (EM) versus equilibrium potential (EION) 1. ionic concentration gradients 2. selective ionic permeabilities 3. at rest, non-gated K channels are open PK high, PNa low 4. K+ efflux separates charge 5. EK+ is most important at rest Hill, ed. 2004. Animal Physiology VM (also called EM) =  {PION x EION} Equivalent circuit of nerve cell membrane (V= I * R); Ohm’s law for flow of charge RM = trans-membrane input resistance ion channels = resistors, RM myelin also affects RM RA (RI) = axoplasmic (internal) resistance RO = external resistance CM = membrane capacitance phospholipid bilayer = capacitance, CM equilibrium potential (amd driving force) = current (I) generator Na/K pump = battery Ion channels - RM present or absent, open or closed Myelin - RM, CM present or absent Exocytosis - CM Diameter of axon - RI Can we explain bio-electrical phenomena with a physics perspective?  Ion channels as conductors of electricity. Ohms law V = iR electromotive “driving” force and conductance IION = gION (VM - EION) Ionic driving force = {VM - EION} Permeability (conductance) PION = gION (and) VION = EION Hyperpolarized membrane (near EK+) low driving force for K+ : K efflux is low, steady, maintaining RP high driving force for Na+ : Na influx is explosive at threshold Ionic driving force and Depolarized membrane (near ENa+) permeability (gION) each factor high driving force for K+ into the membrane potential: low driving force for Na+ VM =  {PION x EION} (slide 11)  Electrotonic (passive) membrane properties - first look Length constant Time constant 1. Record VM 2. Inject I across R VM on, VM off Onset and offset are not immediate in Time (blue) Or across distance (red); V1 vs V2  http://www.nobelprize.org/nobel_prizes/chemistry/ Study of ion channels - X-ray crystallography laureates/2003/mackinnon.jpg Crystals made so that distances between amino acids measured - structure inferred KcsA bacterial K+ channel - Nobel Prize, Roderick MacKinnon exact structure of selectivity filter and pore determined bacterial voltage gated channels crystallized (2008-2011) eukaryotic channels determined by cryo-EM technique Side and top views of the KcsA channel Diagram (side view) of KcsA Study of ion channels - Molecular Biology gene sequences predict association of subunits with cell membrane hydrophobicity plot of gene sequence - H20 - loving / fearing “domains” channel subunits form receptor, pore, and / or accessory units ion channel pore subunits Hydrophilic Hydrophobic hydrophilic hydrophobicity plot AchR (shown) - 5 proteins, 4 genes NaV1.4 - 2 proteins, 2 genes  Using patch clamp to study ion channels Study of ion channels Xenopus oocyte expression system - Electrophysiology Xenopus, African clawed frog. Use oocytes: Have not expressed native ion channels Will express injected, exogenous mRNA coding for channel proteins Will traffic and insert functional channels into membrane Study ion channels with patch clamp electrophysiology Can compare normal channels to those known to be mutated in disease “channelopathies” Na and K channel responses to VM (more in next lecture) Depolarization VM+ VM- Hyperpolarization Na channel: Na IN (open) Then Inactivates With VM-, closes K channel: K OUT (open) With VM-, closes [NOT the K Channel for resting potential] Expression of Na+ channels (only) in frog egg record channels with patch clamp (right) open inactivate Oocytes RNA  Channelopathies can now be studied in expression systems Skeletal muscle Na+ channel: Hyperkalemic Periodic Paralysis Hypokalemic Periodic Paralysis Paramyotonia Congenita Potassium Aggravated Myotonia Brain Na+ channel: Generalized Epilepsy with Febrile Seizures Plus types I and II Severe Myoclonic Epilepsy of Infancy Idiopathic Childhood Epilepsy with Generalized Tonic Clonic Seizures  Ion channels as conductors of electricity revisited. Ohms law V = iR obeyed, or not obeyed. Ohmic channels non-voltage-gated leak channels Rectifying channels ligand-gated channels (non- ohmic) voltage-gated Ohmic channels Voltage-gated sufficient for channels “Passive”, or required for “Graded” response action potential (AP) What is an AP? Action Potentials Why? To carry information rapidly over distance, in axons, and in muscle Frequency coding - intensity of AP Frequency denotes the information Recruitment - number of axons carrying AP denotes information Place Code - identity of axon carrying the AP denotes the information How? Voltage gated Na, K, Ca channels Rapid, + or - feedback faithful propagation of AP over distance Next time. How does AP work?