Bioelectricity and Biophotonics Engineering Past Lecture Notes PDF

WSC331 Bioelectricity and Biophotonics Engineering Felipe Iza P 3G Plasma and Pulsed Power Group Loughborough University, U.K....

WSC331 Bioelectricity and Biophotonics Engineering Felipe Iza P 3G Plasma and Pulsed Power Group Loughborough University, U.K. Slide Set Bioelec 7 [email protected] http://www.lboro.ac.uk/departments/meme/staff/felipe-iza 1 Recap of previous lecture Interior / Cytoplasm Im + IC Ik INa ICl Vm gk gNa gCl Cm Ek ENa ECl - Extracellular medium 2  Concepts  Nernst Potential: Equilibrium C RT  p   e transmembrane potential for a single E p  Z F ln Ci  p  p ion.  Resting Potential: Equilibrium (Itotal=0) Vm   gpEp transmembrane potential. Weighted average of Nernst potentials.  gp  Donnan equilibrium: All permeable CeK CeNa CiCl ions are individually in equilibrium. i  i  e C K C Na CCl  Quasineutrality: Concentrations of anions and cations in a solution are equal. 3 Today’s lecture  Ion Channels  Structure  Biophysical methods: Voltage clamp Patch clamp  Conductance measurements  Channel gating 4 Ion Channels - Historical perspective Rapidly moving field…  1950: The concept of ion channels emerged  1976: Observation of the behaviour of individual channels  1982: Determination of the structure of the first protein channel  200x: ~Thousand of scientific papers per year 5  The cell membrane e Transmembrane potential Vm=i - e +/-100mV i Im Phospholipid bilayer + proteins, channels, pumps,… Ion channels: Selective and gated!! 6  Ion Channels: Structure  Ion channels are pore- forming proteins that allow the flow of ions.  Because of their small size and the difficulty of crystallizing integral membrane proteins for X-ray analysis, we do not know how channels "look like."  Typically they consist of several subunits that enclose a water-filled pore Crystal structure of the CorA Mg2+ transporter Vladimir V. Lunin et al., Nature 440, 833-837 (6 April 2006) 7  Ion Channels: Structure - Genetics  An increasingly important technique for investigating channels is based on gene cloning methods  sequence of amino acids.  Although the primary structure of many channels has now been determined, the rules for deducing the secondary and tertiary structure are not known. Educated guesses on folding can be made, however.  E.g. a run of ~20 hydrophobic amino acids would extend across the membrane. Image from IUPHAR Compendium of Voltage-Gated Ion Channels 2005 8 Classification of ion channels  Global view of the 143 members of the structurally related ion channel genes highlights seven groups of ion channel families and their membrane topologies. F. H. Yu and W. A. Catterall, Sci. STKE, , DOI:10.1126/stke.2532004re15. 9 Ion Channels: Structure  Size > membrane  Non-polar parts embedded in the membrane  Variable cross-section  Selectivity is not simply a steric property (e.g. K channels let pass K+ at a rate 104 greater than Na+ even though Na is smaller).  Some parts contain dipoles Voltage sensitive elements Selectivity & Gating  Gating is mediated by conformal changes induced by Efields and/or ligands Malmivuo & Plonsey, Bioelectromagnetism: Principles and Applications of Bioelectric and Biomagnetic Fields, 2012. 10  Channel conductance  How can we Interior / Cytoplasm determine the channel Im conductance? + IC Ik INa ICl Vm gk gNa gCl Cm Ek ENa ECl - Extracellular medium 11  Voltage clamp  Technique to measure ion currents while holding the transmembrane potential constant  Insights into the channels conductivity  The basic idea: Interior / Cytoplasm Im dVm + IC I m Cm   I ion Ik INa ICl dt gk gNa gCl Vm Cm Fix Vm  I m  I ion Ek ENa ECl - Extracellular medium Voltage clamp measurement: Measure Im at different Vm 12  Voltage clamp  In an ideal world VDC=Vm  All that we need is a DC power supply and amp- meter R Interior / Cytoplasm + Im IC Ik INa ICl VDC Vm gk gNa gCl Cm Ek ENa ECl - Extracellular medium  In reality… R>0  Vm is not VDC!!  Sense Vm and introduce feedback loop 13 Voltage clamp (I)  The membrane voltage, Vm, is measured by an amplifier using an internal recording electrode inserted in the cell, and an external reference electrode. 14 Voltage Clamp (II)  The desired membrane voltage, Vc, is set by the investigator and the difference between Vm and Vc is used to generate a difference signal.  The voltage clamp amplifier generates a signal that is proportional to the difference between Vm and Vc. 15 Voltage Clamp (III)  The difference signal is used by the voltage clamp amplifier to generate a current that is injected into the cell via the current-passing electrode.  This feedback circuit keeps Vm as close to Vc as possible and operates virtually instantaneously. 16 Voltage Clamp (IV)  The current required to keep Vm equal to Vc is measured & recorded.  This current is the ion flux across ion channels as voltage-gated channels open & close. 17  Voltage clamp Vm ENa? EK? Vr? Direction of Na+ and K+ currents? ENa Time Vm1 Vr EK  K+ current: Influx or efflux?  Na+ current: Influx or efflux? 18 Voltage Clamp: Current trace K+ Na+ 19 Voltage Clamp: Current trace More on this in coming lectures 20  Patch clamp  Limitation of voltage clamp technique: we control many channels and of different types at the same time. Not all the channels experience the same transmembrane potential unless special measures are in place.  Can we look at one channel at a time?  Patch clamp  Patch clamp looks a small area of the membrane (m2) rather than the whole cell.  Challenge: Smaller currents!!! 21 Patch Clamp Four configurations:  Cell-attached: Disturbs least the structure and environment of the cell membrane. Channels in normal environment!  Whole cell: Similar to the voltage clamp except that it is suitable for small cells  Outside-out: Well suited to examine ionic channels controlled by externally located receptors.  Inside-out: Well suited to examine the influence of intracellular components in the ionic channels. 22  Patch clamp: Current traces  Patch-clamp current recordings have discontinuities that reflect the opening and closing of individual channels.  Channels have typically 2 states: open and close  Time in each state varies randomly with a given probability of being open and close. Registration of the flow of current through a single ion channel at the neuromuscular endplate of frog muscle fiber with patch clamp method. (From Sakmann and Neher, 1984.) 23 Single-channel conductance  The conductance may depend on the current (not constant value!) 24  Single-channel conductance Channel  (pS) Channels/m2 Sodium Different electrolytes inside/outside Squid giant 4 330 axon Frog node 6-8 400-2000 Rat node 14.5 700 Bovine 17 1.5-1.0 Same electrolytes chromaffin inside/outside Potassium Squid giant 12 30 axon  Macroscopic conductance Frog node 2.7-4.6 570-960 = conductance of the Frog skeletal 15 30 channel x number of Mammalian 130-240 - channels BK 25 Channel gating  Activation / Inactivation: Conformational changes due to:  Changes in the transmembrane potential  Force on charged particles within the membrane.  Binding with Ligands  Inactivation “visualizations” (subject to certain degree of guess work!)  Chain ball (see picture)  Swinging gate 26 Today’s lecture  Ion Channels  Structure  Biophysical methods: Voltage clamp Patch clamp  Conductance measurements  Channel gating 27 Next Lecture  Ion Channels  Link between microscopic and macroscopic quantities  Macroscopic model: Dynamics of a First order system  Hodgkin-Huxley model:  K and Na channels 28