BIO 3350 Lecture 4 Action Potentials PDF

CHAPTER 4: ACTION POTENTIAL The ionic basis of action potentials The sequence of channel openings and closings that generate each action potential How scientists can study the ionic current and the channel function of neurons Readings Bear, Chapter 4...

CHAPTER 4: ACTION POTENTIAL The ionic basis of action potentials The sequence of channel openings and closings that generate each action potential How scientists can study the ionic current and the channel function of neurons Readings Bear, Chapter 4 1  COMMUNICATION IN THE NERVOUS SYSTEM Sensory Neural Interpreta- stimulus code tion/Action Action Activation of potential cutaneous Flexor receptors post-synaptic withdrawal associated with potential reflex intense deformations ??? 2  INTRODUCTION Action potentials Transfers information over long distances … 0.1 mm to 1 m … Neural Code : frequency and discharge pattern ? Synonyms: Spike, nervous impulse, nervous influx, discharge 3 PHASES OF ACTION POTENTIALSS Phases: Resting potential 1. Rising phase (depolarisation) 2. Overshoot 3. Falling phase (repolarisation) 4. Hyperpolarisation 1. Absolute Refractory period 2. Relative Refractory period 4  GENERATION OF ACTION POTENTIAL Caused by a depolarisation of the membrane beyond of a threshold Electrical, chemical or mechanical stimulation « All or nothing » Chain reaction Feedforward loop OPENING OF CHANNELS PERMEABLE TO NA+ 5  GENERATION OF ACTION POTENTIAL Artificial injection of current in a neuron by using a micro-electrode Generation of action potentials 6  GENERATION OF ACTION POTENTIAL Frequency of discharge depends of the amplitude of injected current 1. stimulation too weak… … does not reach action potential threshold 2. Just above the threshold… … a few action potentials 3. Stronger stimulation … … increase of the frequency of discharge 7 MECHANISM OF GENERATION 8  ACTION POTENTIAL: EXPERIMENT OF HODGKIN + HUXLEY Alan Andrew Hodgkin Huxley 9  ACTION POTENTIAL: EXPERIMENT OF HODGKIN + HUXLEY Studied the giant axon of the squid Inserted a metal filament the length of the axon Determined the importance of the permeabilities of Na+ and K+ Made a mathematical model 10  SIMPLE MODEL OF AN ACTION POTENTIAL Membrane potential a) Rest: A few K+ channels open gK >> gNa Vm=-65mV EXTRACELLULAR SPACE K+ Na+ 11 CYTOSOL K+ Na+  SIMPLE MODEL OF AN ACTION POTENTIAL b) Depolarisation crossing the threshold: Rapid opening of Na+ Membrane potential channels gK > gNa Vm repolarize EXTRACELLULAR SPACE K+ Na+ 13 +  SIMPLE MODEL OF AN ACTION POTENTIAL Membrane potential d) Return to rest: Some K+ channels open gK >> gNa Return to rest Vm=-65mV EXTRACELLULAR SPACE K+ Na+ 14 +  ACTION POTENTIALS IN REALITY Major problem with this simple model How do Na+ channels close at depolarized Vm and stay closed long enough for K+ channels to repolarize the membrane If Na+ channels reopen, this would Na+ channels happen must stay close for K+ channels to repolarize 15  ACTION POTENTIAL IN REALITY The experiments of Hodgkin and Huxley show that action potentials are more complicated Require that certain channels inactivated. I.e. they become incapable of passing through current independant of membrane potential. How can this mechanism of inactivation been verified? 16  MINOR(?) ASIDE CELLULAR ELECTROPHYSIOLOGY Techniques to observe the electrical activity of a neuron For exemple, we would like to determine which currents pass the membrane during an action potential 17  ELECTROPHYSIOLOGY Recording electrode Ielectrode Circuit for Im measurement Rm vm electrode in the bath In the lab, we can measure electrical signals. Vm = Rm x Im (Ohm’s law)  ELECTROPHYSIOLOGY Recording electrode Ielectrode Circuit for Im measurement Rm vm electrode in the bath On the other hand, we can only measure either of (Vm or Im) at a time Vm = Rm x Im (Ohm's Law)  ELECTROPHYSIOLOGY Recording electrode Ielectrode Circuit for Im measurement Rm vm electrode in the bath By using our electrode, we can either 1. Impose a current and measure the Vm that results 2. Impose a voltage and measure the Im that results  IMPOSED VOLTAGE VS IMPOSED CURRENT recording clamp Current-clamp Useful to oberve the behaviour of a neuron by Vm V=RxI Useful to observe the Voltage-clamp properties of Ion channel by monitoring their conductances clamp recording 21  CURRENT-CLAMP Let us measure Vm by injecting currents through an electrode (Ielec) Recording (V) Recording electrode Ielectrode Circuit for Im measuremen t Rm vm electrode in the bath STIMULATION (I)  VOLTAGE-CLAMP Voltage-clamp To measure Im at a desired Vm, we use an electronic circuit that can fix (or “clamp”) Vm at a value (Vclamp), and then measure the current that the circuit must inject to maintain Vm at Vclamp. Ielectrode Voltage-clamp Im circuit Rm vm  VOLTAGE-CLAMP The voltage-clamp circuit operates using the following logic 1. Inject a Ielectrode and measure Vm 2. If Vm is different than Vclamp, ajust Ielectrode 3. Repeat steps 1 and 2 iteratively until Vm = Vclamp Ielectrode Voltage-clamp Im circuit Rm vm  VOLTAGE-CLAMP By using the voltage-clamp, we A. Voltage-clamp jump of -65 mV to 0 mV can measure the Na+ and K+ current activated when Vm goes from rest to the peak of an B. Im (composed of IK and of INa) action potential Im (mA/cm2) The K+ current (IK) is activated by the depolarisation and it is sustained at throughout the C. K+ current (IK) – after block of INa by TTX depolarisation The Na+ current (INa) is activated by the depolarisation D. Na+ current (INa) – after block of IK by TEA however it is not sustained throughout the depolarisation. This current inactivates. Can we determine why the Na+ current (INa) seems to inactivate after the depolarisation? 26 PATCH-CLAMPING Pipette apposed to the membrane Extract a portion of the membrane Impose a voltage Allows you to record from a single ion channel at a time 27  Leak channels Vm=V1 movement of the “gate” does NOT depend on Vm Vm=V2 ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- open closed Voltage-gated channels movement of the “gate” does depend on Vm Vm=V1 ++++ ++++++++++++++ ---- ---------------  Leak channels Vm=V1 movement of the “gate” does NOT depend on Vm Vm=V2 ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- open closed Voltage-gated channels movement of the “gate” does depend on Vm Vm=V2 Vm=V1 ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- When the activation gate of a channel opens… ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- closed → open ACTIVATION When the activation gate of a channel opens… ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- closed → open ACTIVATION opposite process… ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- open → closed DEACTIVATION  A channel with an activation gate can be either opened or closed ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- C O  Some v-gated channels have both activation and inactivation gates ++++ ++++++++++++++ ---- --------------- both gates need to be open for the channel to pass current (channel in open state) ++++ ++++++++++++++ ---- --------------- One gate in the closed position is sufficient to prevent current flow ++++ ++++++++++++++ closed ---- --------------- ++++ ++++++++++++++ inactivated ---- ---------------  Voltage can drive changes in channel conformation ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- ---- --------------- Closed Open Deactivated (activation gate closed) Activated (activation gate open) Not inactivated (inactivation gate open) Not inactivated (inactivation gate open) ++++ ++++++++++++++ ++++ ++++++++++++++ ---- --------------- Inactivated Deactivated (activation gate open) Not inactivated (inactivation gate closed)  Voltage can drive changes in channel conformation ++++ ++++++++++++++ ++++ ++++++++++++++ ++++ ++++++++++++++ 1 ---- --------------- ---- --------------- Closed Open ACTIVATION (1) depolarization 3 ++++ ++++ ++++++++++++++ ++++++++++++++ 2 ---- --------------- RECOVERY FROM INACTIVATION (3) INACTIVATION (2) repolarization Inactivated maintained depolarization  An inactivating Na+ channel activated by depolarization Vcom -40 mV -80 mV Notice zero current at -80 mV (channels are closed, i.e. activation gate closed). Channels open with a delay after the voltage is changed to -40 mV. Current is driven both by channels being activated (open) by voltage and by the driving force. Channel inactivate (close) after some time at -40 mV. Typical sodium channel with both activation average current and inactivation gates.  RECORDING OF Na+ CHANNELS Rapid opening of channels to Na+ Very short delay (< 1 ms) Conformational change to open the pore Stays open for about 1 ms Unitary conductance Rapid closing Mechanism of inactivation 3 « Ball and chain » Potential must be repolarised for re- activation4 Na+ channels must be inactivated after action potential Absolute refractory period 38  PHASES OF ACTION POTENTIALS By using patch-clamping, we can measure currents of Na+ channels individually during an action potential This allows the measurement of the Na+ current that enters Conclusion: The Na+ current only enters during the Rising phase 39  A non-inactivating K+ channel activated by depolarization Vcom +50 mV -100 mV Notice zero current at -100 mV (channels are closed, i.e. activation gate closed). Channels open with a delay after the voltage is changed to +50 mV. Current is driven both by channels being activated (open) by voltage and by the driving force The average current has the same kinetics of the total membrane current, but smaller amplitude. Typical potassium channel (delayed rectifier) with only activation gate. average current  PHASES OF ACTION POTENTIALS The recording of K+ currents of individual channels is also possible This allows us to measure the K+ current entering Conclusion: The K+ current entres at the end of the Rising phase and peaks at the start of the Falling phase 41  K+ CHANNELS VS Na+ CHANNELS Dependance to voltage The 2: open in response to a depolarisation K+ channels open more slowly than Na+ channels Only Na+ channels inactivate Delayed rectifier K+ condutance serves to rectify or reset Vm Necessary to free Na+ channels from their inactivations Structure K+ channels: 4 subunits Na+ channels: 4 domains 42  GENERATION OF AN ACTION POTENTIAL Membrane potential a) At rest: Some K+ channels open. All Na+ channels are closed gK >> gNa Vm=-65mV EXTRACELLULAR SPACE K+ Na+ 43 CYTOSOL K+ Na+  GENERATION OF AN ACTION POTENTIAL Membrane potential b) Depolarization crossing the threshold: Opening of a small number of Na+ channels Vm depolarizes EXTRACELLULAR SPACE K+ Na+ 44 CYTOSOL K+ Na+  GENERATION OF AN ACTION POTENTIAL Membrane potential c) Rising phase: Rapid opening of all Na+ channels gK > gNa Vm repolarize EXTRACELLULAR SPACE K+ Na+ 46 CYTOSOL K+ Na+  GENERATION OF AN ACTION POTENTIAL Membrane potential e) Falling phase Na+ channels stay inactivated Opening of all K+ channels gK >> gNa Vm repolarize EXTRACELLULAR SPACE K+ Na+ 47 CYTOSOL K+ Na+  GENERATION OF AN ACTION POTENTIAL Membrane potential f) Refractory period: Some K+ channels open Na+ channels stay inactivated which prevents the initiation of another action potential EXTRACELLULAR SPACE K+ Na+ 48 CYTOSOL K+ Na+  GENERATION OF AN ACTION POTENTIAL Membrane potential g) Return to rest: The inactivation of Na+ channels is terminated The cell can initiate another action potential EXTRACELLULAR SPACE K+ Na+ 49 CYTOSOL K+ Na+ PROPAGATION OF ACTION POTENTIALS 50 PROPAGATION OF ACTION POTENTIALS Orthodromic Action potential travels towards the nerve terminals (red arrows) Antidromic From nerve terminals towards the dendrites (blue arrow) Duration of an action potential: 2ms Typical conduction velocity: 10m/s 51  FACTORS INFLUENCING CONDUCTION VELOCITY: SALTATORY CONDUCTION Myelin Schwann cells (PNS) Oligodendrocytes (CNS) Multiple lipid layers around the axon Very few Na+ channels Prevents the leakage of ions Nodes of Ranvier Uninsulated High density of Na+ channels Allows the passage of ions 52  FACTORS INFLUENCING CONDUCTION VELOCITY Electrotonic conduction (« passive ») underneath myelin Passive diffusion of Na+ ions in the longitudinal axis of the axon Myelin prevents loss of Na+ ions out of the axon Active conduction (with ion channels) at nodes of Ranvier Action potentials with Na+ channels 53  ACTION POTENTIALS, AXONS AND DENDRITES Initiation zone of action potentials In sensory neurons Nerve terminals (dendritic extremities) Other neurones Axon hillock 54