Electrophysiological techniques

• ELECTROPHYSIOLOGY BASICS

  • Ions are charged (flow of ions =current)
  • sticking an electrode inside the cell and one in the bathing solution --> current voltage can be measured between sample and surrounding samples
  • ADVANTAGES:

  1. can measure events on fast timescales
  2. can measure activity of single ion channels
  3. spatial resolutions can be good
  4. can tell us how channels work
• THREE MAIN ELECTROPHYSIOLOGY TECHNIQUES:

  1. extracellular
  2. intracellular
  3. patch
• EXTRACELLULAR TECHNIQUE

  • easy 
  • not very specific
  • electrode is placed outside, but close to it
  • tells us activity of group of cells 
  • comes through eeg --> electroencephalogram

  1. electrodes placed on scalp and records brain activity in different regions 
  2. localize brain region where the problem is 

  • comes through ecg --> electrocardiogram
• INTRACELLULAR TECHNIQUE

  • hard
  • specific
  • hard blast electrode is poked through the cell
  • can tell potential different and current
  • good for large cells but are hard to do
  • requires specialized equiptment 
  • sharp electrode is poked through a cell and can record action potential
• PATCH

  • very hard and specific
  • can give info about activity of single channels
  • most important techniques in neuroscience
  • invented in 1970-1980 by sakmann and neher
  • collection of 4-5 types of configuration
  • allows recording of single channels

  1. cell attached,  outside-out, inside-out

  • allows recordings of many channels

  1. whole cell, perforated patch

  • fine glass electrode filled with conducting solution connected to a wire to a surface of the cell -->needs to be stable and an electrically clean recording

  1. no vibrations and no outside noise
• WHOLE CELL RECORDING

  • cell-attached is the starting point
  • and futher suction disrupts the membrane
  • inside of the electrode is continuous to the inside of the cell
  • if drugs are applied --> can observe the conductance of any channels that are activated by the drug across the whole cell 
  •  used in drug screening and most common
  • similar to perforated --> antibiotic is added which acts as a wide diameter pore through the membrane (allows electrical continuity)
• INSIDE-OUT MEMBRANE

  • membrane is inside out --> faces the bath
  • start in cell-attached mode 
  • pull the membrane away from the cell --> membrane is trapped
  • take electrode out of the bath --> encourage ends to seal onto electrode 
  • put it back in the bath --> inner membrane faces the bath somtuin
  • important ins investigating regulation of a channel or receptor by apply something to the inside face
  • hard to change whats in the solution
• OUTSIDE-OUT MEMBRANE

  • faces the tip
  • starts with cell-attached patch --> whole cell
  • rips a piece of membrane off --> keep membrane in the bath --> encourage the ends to seal 
  • hard recording to set up 

  
• APPLICATIONS

  • Cell-attached:

  1. to record currents through a limited number of active channels at the cell surface
  2. good for looking at single channel currents in response to regulations of channels by cells

  • Whole-cell:

  1. to record currents through active channels in the whole cell
  2. good for looking at cell currents in response to drugs added from the outside or regulation of channel by cells --> can change solutions fairly rapidly
• APPLICATIONS cont.

  • Inside out:

  1. to record current through a single active channel away from the cell
  2.  good for looking at agents that modulate channel by working at its intracellular face

  • Outside out:

  1. to record currents through a single active channel away from the cell
  2. good for looking at agents that modulate channel by working at its ectracellular face
  3. can change solution rapidly
• SINGLE CHANNEL PROPERTIES OF ION CHANNEL

  • channels are molecular machines --> capable of transitioning between different states
  • activation mechanism of a simple channel

  1. k-1 --> closing rate constant --> stability of open state (big value --> brief opening)
  2. k+1 --> opening state constant  stabilitie of closed state (big value --> brief closing)

  • pattern of activity is fairly random 
  • mid point for closed time  1/k+1
  • mid point for open time  1/k-1
  • studying patterns of opening nd closing can tell us about the activation mechanism
• SINGLE CHANNEL PORE PROPERTIES

  • bit of current --> ions moving
  • current = ions per second (dependent on [ion], voltage and the pore itself)
  • high conductance = bigger sized single-channel event
  • low conductance = smaller sized single-channel event
  • opening and closing is the same length
  • if a mutant channel saw a change in the cpbnductance --> we can determine what the mutation is
• TWO-ELECTRODE VOLTAGE CLAMP

  • involves putting two electrodes in the cell

  1. voltage electrode --> membrane potential
  2. current electrode --> injects current to maintain the clamp