Voltage gated ion channels

Voltage gated ion channels

1. A rising phase of an action potential is when the level of sodium rises, Repolarisation is when the potassium leaves the cell

2. PATCH CLAMPING: Measures the current of a single channel using a microelectrode

   • Can see the channel opening and closing and so can see how the action potential affects permeability of the channel
     • Depolarization increases the probability that the channel so the channel may not always open at the exact time but it will be approximatly in this time
       • 1. Na+ channels open 1st due to depolarisation, 2. Na+ channels are then inactivated during depolarisation, 3.K+ channels open later than Na+ channels but dont become inactivated
   • For K+ you can see that the channel open and close more and this happens over a longer period of time

3. Toxins that block ion channels

   • Na+ blockers
     • TTX (Tetradotoxin)
       • It is found in puffer fish
       • STUDY: Voltage clamping a squid axon in TTX blocks inward current caused by Na+ but outward current is unaffected
     • SATITOXIN(STX)
   • K+ blockers
     • TEA (Tetrylethylammonium)
       • Study: Blocks outward current when voltage clamping
     • 4- Aminopyridine (4-AP)

4. Na+ Voltage gated channels

   • STUDY: The electric eel E.Electricus has organ that is enriched in Na+ channels, TTX purified the channels to find a protein known as the a-subunit which has 4 domains
     • Each domain has 6 transmembrane helices ( made of hydrophobic amino acids so they can span the membrane)
       • The picture below shows the a subunit
     • There are also 2 b subunits in the mammalian Na+ channnels but the a subunit makes up the pore
   • Structure: 1. Activation/Voltage sensitivity 2.Selective permeability 3. Inactivation
     • S4 (The 4th helix) : It is the voltage sensor, it is arginine and Lysine rich (Positively charged) this affects voltage sensitivity
       • STUDY:If the amino acids are mutated to acidic or basic ones this changes the voltage sensitivity
       • It is thought that the S4 region moves outward at depolarization to from a close to open conformation
       • S4 doesn't completly span the membrane in K+ channels and most likely in Na+ , Found by studying bacteria A.pernix. It acts as a paddle
     • Selective Permeability: Between S5 and S6 there is a region called P which forms the lining of the pore
       • The pore lining has amino acids DEKA
         • STUDY: DEKA was mutated to DEDA or DEKD which had different properties and this converted it into a voltage gated CA2+ channel
     • Inactivation: Between DOMAIN 3 and 4 there is a loop that folds over the pore , the 3 amino acid are I, F and M
     • Na+,Ca2+ and K+ voltage gated channels are thought to be related due to their similar structure
       • Na+ and Ca2+ Voltage gated channels are the most similar
       • The K+ channel also has 4 domains but different subunits
       • Must have a common ancestor
         • There must have originally been one domain and then duplications resulted in the 4 domains
           • This predicts that helices 1 + 3 and 2+4 would be more similar as they were originally the same and this is true
         • Within each domain the Na+ and Ca2+ are similar than the other domains
           • Ca2+ channels came first as there are different types of it and the T type channel evolved to the Na+ channel
   • There are 9 different Na+ voltage gated ion channels
   • CASE STUDY: CONGENITAL INABILITY TO EXPERIANCE PAIN
     • The Na+ channel SCN9A was mutated, it usually carries the pain signal from the peripheral to CNS
       • Insesitivity to pain is caused by the closed state of the Nav1.7 inhibitor, studied tarantula venom

5. VOLTAGE GATED K+ CHANNELS

   • 1. DELAYED RECTIFIER
     • Can be inactivated, blocked by 4AP and dont have a delay
   • 2. Ka/FAST/EARY
     • Respond with a delay, they don't inactivate , are blocked by TEA and mediate falling phase
   • STUDY: A mutant drospholia has a SHAKER gene (legs shake even under anesthesia ), Ka type currents are affected by this-dont function
     • The shaker gene has 616 amino acids and transmembrane helices (like a Na+ domain)
   • There are 4 voltage gated K+ channels in Drospholia which have different properties
     • SHAKER
     • SHAL
     • SHAB
     • SHAW
       • In shaw there is no inactivation, each of the genes have different rates of activation and inactivation
     • There are more equivelant mammalian genes
     • The channels are diverse because the different subunits can make different combinations and different gene products are made by mRNA splicing
       • They mediate the falling phase
       • stop firing of action potientials when there is low depolarisation
       • Determine the shape of action potentials
   • There are K+ channels that are NOT voltage sensitive, only have 2 domains instead of 4
     • Study: S.Lividans (bacteria ) have this
     • The selectivity filter is made up of glycine, tyrosine and glycine (GYG) which favours passage of K+
   • INACTIVATION: not the same as voltage gated Na+ channels due to the different subunits
     • There is a ball and chain mechanism where the n terminal blocks the pore, one of the 4 terminals can do this

6. Ca2+ channels

   • 1L-type-involved in muscle contractions, they last for long and are blocked by dihydropyridines, activated at high voltage
   • 2. N-,P,Q-,R type : They are involved in neurotransmitter release, are NOT blocked by dihydropyridine are activated at high voltage
   • 3. T type: Activated at low voltage, last for a short time and are NOT blocked by dihydropyridines
     • The T type has a similar mechanism of inactivation as Na+
   • There are 10 different genes of voltage gated Ca2+ channels
   • Selective permeability: The pore lining has 4 glutamates (EEEE) - negatively charged
     • Normally blocks Na+ but if calcium is low it will allow it through
   • HVA inactivation is slower which is triggered by a rise in inercellular ca2+ which is mediated by Camodulin

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