• glass tubing can be heated and pulled to fabricate micropipette w/ extremely small tip diameter and hole in tip that is continuous w/ lumen of glass tubing
• can be filled w/ ionic solution that conducts electricity to provide electrical path all the way to tip of microelectrode
• glass is excellent insulator allowing us to measure only voltage at the tip of the microelectrode
• glass seals well to cell membranes and its ability to be used in making small-diameter tips allows us to penetrate small neurons w/out causing significant damage

   

• primarily leakiness of the cell membrane
  • hydrophobic chemistry of cell membrane imparted by fatty acid tails prevents passage of ions
  • presence of numerous ion channels allows flow of ions into and out of the membrane
    • greater number of open ion channels = greater number of resistors in parallel = lower equivalent resistance
  • the ability of the cell to open, close, insert, or delete these channels allows the cell to adjust its resistance
• important trend: as cell diameter increases resistance...
  • decreases as increasing membrane surface area means more resistors that are in parallel leading to a lower equivalent resistance of the membrane

• lipid bilayer is extremely thin (on the order of 5nm)
  • as we know, the distance between plates is inversely related to capacitance
  • therefore, this extremely small distance between monolayers of the lipid bilayer makes it an outstanding conductor (1 uF/cm²)
• important trend: as cell diameter increases, capacitance...
  • also increases due to the increase in the surface area of the bilayer
  • as we know, area of the plate and capacitance are directly related, so more membrane = higher capacitance

• Biological membranes are very good capacitors because they are extremely thin (about 5 nm)

  resistors separating two conductors, the intracellular and extracellular solutions of the cell.

  Charges on either side of the membrane can approach each other very closely because the

  membrane is so thin, allowing many charges to be stored for a given voltage.

• longtime understanding that cell membranes were impermeable to dyes as the cell injected into one dye did not escape to others
• Overton ran studies determining what types of molecules could cross membrane determining that it must be made of lipids
• people postulated the presence of pores to account for permeability to ionic substances (how else could they efficiently cross nonpolar lipid membrane)
• Experiments w/ red blood cells
  • Langmuir Trough
    • calculations using assumed monolayer and average SA of RBC's were half of data actually collected through Langmuir trough
    • this indicated that there must be a bilayer

• biochemistry - isolate and purify protein and study its chemical properties
  • characterizing protein requires immense amounts of isolated, pure protein which can be very difficult to extract as it is surrounded by many other channels 
    • electric eels carry extremely large number of sodium ion channels w/in electric organ allowing us to more easily isolate and purify protein for study
• molecular bio - determine amino acid sequence through cloning and sequencing 
• electrophys - study effect of channel w/in functional environment (use antagonists, remove channel, etc. and see what happens)
• structural tech. - x-ray crystallography can be used to determine general tertiary/quaternary structure

• Transmembrane regions of channel proteins must be rich in hydrophobic amino acids while extra-/intracellular regions must be rich in hydrophilic amino acids
• this can be determined using a hydropathy/hydrophobicity plot
  • we know that transmembrane regions commonly contain alpha-helices of ~20aa w/ hydrophobic R groups pointing out from center
    • we can look for these in primary sequence using moving 20aa window to find average hydrophobicity at certain points
  • by looking for peaks followed by dips into the hydrophilic negative region of the graph, we can determine potential locations of transmembrane regions
  • by identifying repeating patterns of peaks and dips, we can identify transmembrane domains of the protein
• See Slides 11 and 12

• channel is made up of 4 domains
• all 4 staves are one protein (α-subunit)
  • staves are the individual subunits making up the pore opening
  • in the image below, the staves are the greenish grey subunits
  • in the sodium ion channel, the 4 staves are the 4 domains of the α-subunit not including the p-loops

[image]

[image]

• express the protein
  • express ion channel in cell w/out (or w/ minimal) other ion channels that could interfere w/ identification of desired protein
    • oocytes of the xenopus laevis 
  • synthesize cDNA to make cRNA
  • inject the cRNA into the ideal cell choice
• test channel electrophysiologically
  • use oocyte modified to express channel
  • do a voltage clamp experiment to confirm the presence of the channel and that it exhibits the correct properties
    • e.g.: if i am testing voltage-gated channel, what happens if I depolarize the cell above certain threshold? we should see action potential 

• can be used to identify what region of protein lines channel pore
• Substituted Cysteine Accessibility Method
  • mutate one amino acid residue at a time to a cysteine residue
  • measure current before and after exposure of oocyte to hydrophilic reagent MTSEA
    • reacts selectively w/ sulfhydryl groups to form disulfide bonds when sulfhydryl group is exposed to water (won't interact w/ transmembrane or internal residues)
  • if mutated aa is exposed to pore, then its mutation and interaction w/ MTSEA will result in blockage of current

• specific locations of protein contributing to this loop can be determined through SCAM
• these are sections of protein that contribute to the channel pore
• found between S5 and S6 of each channel stave
• aa's in p-loop are what interact directly w/ the ion that passes through
  • this is likely difference between different types of ion channels (like K⁺ vs. Na⁺ vs. Ca⁺⁺)

• very similar structure to sodium channel w/ 4 staves each of 6 segments (S1-6)
• the big difference is that the potassium channel is tetramer w/ each stave being a different peptide (so 4 small alpha vs. just 1 large alpha subunit)
  • labeled as I, II, III, IV in this image

[image]

• extremely similar to sodium channel as it is one long alpha subunit (w/ small alpha-2 subunit)
• the alpha-1 subunit is made up of 4 staves each of 6 segments (S1-6)

[image]

• Inactivation gate
  • a semi-unique feature of voltage-activated sodium channels that make it immensely more difficult to trigger another action potential before falling below threshold
  • cytoplasmic portion of protein located on the linker between domains 3 and 4
  • returning to threshold causes inactivation gate to open making cell go back to deactivated confirmation

[image]

• subunits other than alpha (or alpha-1) of voltage-activated ion channels
• they are not necessary for channel functioning
• typical have sites for chemical modification that can alter properties of the channel like conductance 
• beta units in the voltage-activated sodium channel

[image]

• important as it provides us with a way of determining protein structure
• a beam of x-rays is aimed at a single protein crystal causing the beam to scatter creating a diffraction pattern
• process is repeated as crystal rotates
• each time the strength and angle of the beams are recorded providing data that can be turned into an electron density map followed by an atomic model
  • each spot is a reflection of an X-ray from evenly spaced planes w/in crystal
  • provides diffraction data that is used to determine mean chemical bond lengths and angles w/ extreme accuracy
  • allows you to build electron density map

[image]

• commonly determined by comparing aa sequences of channels accepting the same type of ion
  • for example, comparing a variety of K⁺ channels and seeing the repetition of the TVGYG sequence indicating its importance
• how pores are designed to accept particular ions
• the selectivity filter is for channels is always found contained w/in the p-loop
  • selectivity can be based on pore size, charge of amino acids in pore
  • commonly based on ability of pore to strip waters off hydrated substrates
  • K⁺ channel as example
    • hydrated ions too large for channels
    • waters must be stripped to allow for entry
    • TVGYG sequence amino acid carbonyl oxygens can do this job
    • smaller ions like Na⁺ remain unable to pass as their electrostatic interactions are stronger and waters are not removed as easily
      • 2 waters removed from K⁺ as opposed to the 1 removed from Na⁺
      • diameter of pore in Na⁺ channels is actually larger for this reason, but will not pass K⁺ as its geometry does not allow for stabilization of K⁺ ion

• ionic movement through p-loop is very quick
  • how could this be possible if ions do not move with direction
  • this is because multiple ions pass through channel at a time putting them in close proximity
  • electrostatic repulsion pushes these ions quickly through the channel

• theory that nervous system was labyrinth of circulating fluid around body
• at one point it was even thought that their purpose was to keep blood cool due to proximity of nerves to blood supplies
• likened nervous system to cardiovascular system

• Cajal v. Golgi
• Contributions of Cajal
  • identified the two main cell types in the nervous system - glia and neurons
  • used golgi's method to show that neurons were not continuous labyrinth - contributed to neuron doctrine
    • also contributed to dynamic polarity
• Ross Harrison
  • showed that neural processes (dendrites and axon) grow from the cell body when neurons are isolated in culture, axons can extend to target neurons or tissue
• Sanford Palay
  • images synapses and gaps between neurons

• the neuron is the fundamental structural and functional unit of the nervous system
• neurons are discrete cells which are not continuous with other cells
• the neuron is composed of 3 parts – the dendrites, axon and cell body, and
• information flows along the neuron in one direction (from the dendrites to the axon, via the cell body)
  • Termed the principle of dynamic polarity

• many factors can be considered, but it mainly comes down to changing membrane properties (particularly resistance)