CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES,NORBERT JOST, PhD,Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique,G=1/R,Ohms law,Ion channel model,Current clamp,Voltage clamp,Intracellular microelectrode technique,Re Rin Rin = 1012 Ohm,0.1 - 0.2 m,Ag/AgCl 3 M KCl,Re 10 - 40 MOhm,The setup,Organ bath,d: stimulating electrode,e: microelectrode,r: referent electrode,P: preparation,computer,A/D,ingerl,amplifier,Detected signal,P,d,e,r,100 ms,50 mV,0mV,drug,60 min,20-60 min,60 min,0 mV,APA,RP,APD50,APD90,90%,50%,Vmax,Wash-out,Pre-incubation,Two microelectrode voltage clamp,voltage command,holding potential,test potential,The macroscopic sodium current,The voltage-clamp circuit,voltage command,amplifier,Current measure,voltage measure,follow up amplifier,Patch-clamp: the special case of the voltage clamp,Cell,Patch-clamp: the special case of the voltage clamp,(1) Suck a small piece of membrane onto the tip of a glass micropipette ( 1 m in diameter),Cell,(2) “Gigaohm-seal”,R 1 GOhm,Patch-clamp: the special case of the voltage clamp,Cell,(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.,Patch-clamp: the special case of the voltage clamp,closed open,Cell,+ +,Patch-clamp: the special case of the voltage clamp,(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.,closed open open,Cell,(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique.,Patch-clamp: the special case of the voltage clamp,voltage command,10 msec,Properties of individual voltage-dependent sodium channels,Individual channels are either open or closed (no partial openings),Properties of individual voltage-dependent sodium channels,Individual channels are either open or closed (no partial openings) Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current.,The macroscopic sodium current,Properties of individual voltage-dependent sodium channels,Individual channels are either open or closed (no partial openings) Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. Channel opening and closing is variable in duration and latency.,Properties of individual voltage-dependent sodium channels,The macroscopic sodium current,The channels are either in open or closed state. The channel openings are short events when compared with the macroscopic sodium current. The time duration and latency of the channel openings are variable (case sensitive). Might happen to not open at all. The open probability of the channels resembles with that of the macroscopic current.,Properties of individual voltage-dependent sodium channels,The macroscopic sodium current,Summation of 300 recordings,Individual channels are either open or closed (no partial openings) Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. Channel opening and closing is variable in duration and latency. The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. Sometimes an individual channel doesnt open even once.,Summation of 300 recordings,Properties of individual voltage-dependent sodium channels,The macroscopic sodium current,Individual channels are either open or closed (no partial openings) Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current. Channel opening and closing is variable in duration and latency. The overall probability of channel opening is similar to the total sodium current. Look at the sum of the currents from 300 trials. Sometimes an individual channel doesnt open even once. Second openings are rare (because of inactivation),Summation of 300 recordings,Properties of individual voltage-dependent sodium channels,The macroscopic sodium current,Slowly inactivating K current channel (Ram & Dagan, 1987),Individual channels are either open or closed (no partial openings). Sometimes more than one channel is in a