Channel Gating

Question 1

1. describe the main property of voltage gating
2. give 4 examples of voltage gated ion channels
3. describe the main property of ligand gating
4. give 2 examples of ligand gated ion channels.

Answer 1

1. Po changes with Vm
2. Na_v, K_v, Ca_v, CLCK
3. Po changes with ligand binding
4. AcHr, PX2

Question 2

1. What equation is used to show voltage dependent gating?
2. describe the process of how a change in Vm governs Po.
3. How do voltage gated channels detect changes in Vm?

Answer 2

1. Boltzman equation
2. change in Vm induces a conformational change, which either opens or closes the channel
3. they contain voltage sensitive amino acids in the S4 region.

Question 3

1. How was the importance of the S4 region examined. Describe the 2 types of mutations that were made and the amino acid substitutions made in order to do so?
2. In what type of mutant were these experiments performed in? Why?
3. what were used to ensure that the driving force at different Vms were the same. Describe the process of doing this.
4. what was the result of a charge neutralising mutation in the 4th charge in domains 1 and 2?
5. what was the result of a charge neutralising mutation in the 4th charge in domain 3?
6. what was the result of a charge conserving mutation in the 4th charge of domain 3? What does this suggest?
7. What are the overall conclusions of this study?

Answer 3

1. By making charge conserving or charge neutralising mutations in the S4 regions.Charge conserving = Arg > Lys // Lys > ArgCharge neutralising = Arg > Glut // Lys > Glut
2. IMFQ3 mutant. It lacks a ball and chain, therefore eliminates the problem of fast inactivation.
3. TAIL CURRENTS. Cells first subjected to different Vm’s and then the same Vm. If the current is measured immediately after the Vm has been changed, then the Po will still reflect that at the different Vms.
4. positive shift in Vo
5. negative shift in Vo
6. positive shift in Vo. Therefore, it is not just the charge, but the amino acid that is important for Vdep at this residue
7. The 4th charge of S4 domains 1, 2 and 3 are important in the Voltage dependence.

Question 4

1. What governs Vdep inactivation? How does this work?
2. where is the domain that governs Vdep inactivation located in a) Na_v and b) K_v?
3. How was the role of charged residues investigated?
4. What was the result of the above experiment?
5. How were the effects of the amino acid sequence on Vdep activation investigated?
6. what were the results of this study?

Answer 4

1. Ball and Chain mechanism. Ball forms a plug which blocks pore
2. a) an aa plug tethered to 2 chains tethering it to S3 and S4
   b) N terminus
3. 15 charged residues in the III-IV linker region were mutated in blocks of 3
4. No difference in Vdep activation, suggesting that charged residues aren’t important in Vdep inactivation
5. deleted 5 blocks of 10 amino acids from the III-IV linker region
6. deleting the 2nd and 3rd block produced non-functional channelsdeleting the 1st and 4th block abolished fast inactivationdeleting the 5th block had no effect* suggests that amino acids in the 1st and 4th blocks of 10 amino acids are responsible for Vdep inactivation

Question 5

1. What are outward rectifiers?
2. What are inward rectifiers?
3. Name the 3 causes of rectification

Answer 5

1. channels which carry a bigger outward current than inward current
2. channels which carry a bigger inward current than outward current
3. Vdep Po, Vdep block, goldman rectification

Question 6

1. in which channels is goldman rectification seen?
2. what is goldman rectification dependent upon?
3. What is a Vdep block?
4. How was Vdep block studied in Kir channels?
5. What were the results of these experiments?
6. What were the conclusions of these experiments?

Answer 6

1. ALL channels
2. dependent upon EC and IC concentrations
3. in asymmetrical solutions, rectification is seen, due to the presence of something blocking the channel (e.g. Mg blocks Kir channels causing inward rectification)
4. analysing rectification when patches of membranes were excised in an inside out fashion, allowing the [Mg] concentration to be varied
5. when the membrane was excised in an inside out manner, rectification was abolished. The addition of Mg to the internal solution restored this rectification

when [Mg]i is high, rectification is seen; when [Mg]i is low, rectification isnt seen.

6. Mg is the naturally occuring blocking particle causing rectification of Kir channels

Question 7

1. describe the process as to how a ligand can govern Po
2. how many binding sites may a ligand bind to on an ionotropic receptor?

Answer 7

1. ligand binding induces a conformational change in the ion channel causing its opening or closing.
2. 1 or more (some ion channels contain multiple ligand binding sites)

Question 8

Name 4 functional roles of ligand gated ion channels

Answer 8

1. involved in fast chemical synapses
2. nociception
3. paracrine and autocrine signalling
4. mechanosensation (e.g. PX2 is an ATP gated non-selective ion channel. Ligand binding causes a change in intracellular calcium levels in response to muscle tension)

Question 9

1. What ions are Glycine receptors selective for
2. what is the E_i for this ion? Therefore, are they excitatory or inhibitory channels and why?
3. what ions are GABA_A receptors selective for? Therefore does this make them inhibitory or excitatory?
4. what ions are glutamate receptors selective for?
5. What is the nernst potential for the channel? Therefore are they excitatory or inhibitory?
6. What are they activated exogenously by?

Answer 9

1. chloride
2. E=-70mV. channel opening drives the Vm further away from threshold therefore the cell becomes hyperpolarised, and the channels are INHIBITORY
3. chloride. inhibitory
4. non selective
5. 0. Therefore, they are excitatory
6. AMPA, NMDA or Kainate

Question 10

1. what is the relationship between E_cation and Vm for glutamate receptors. Therefore, what currents are produced upon channel opening?
2. what is the releationship between E_Cl and Vm for glycine receptors under a) physiological conditions and b) experimental symmetrical conditions?
3. Therefore what currents are produced upon channel opening under a) physiological conditions and b) experimental symmetrical conditions?

Answer 10

1. Vm is negative to E_cation therefore cations move inward, producing inward EPSCs
2. a) Vm is positive to E_Cl
   b) Vm is negative to E_Cl (E_Cl=0 under these conditions)
3. a) Cl moves inward producing outward EPSCs
   b) Cl moves outward producing inward EPSCs

Question 11

1. Name 2 structures in which glycine receptors are highly expressed
2. describe the subunit composition of the glycine receptor
3. how many subunits need to be mutated for a non-functional ion channel to be produced?

Answer 11

1. brainstem and spinal cord
2. 3α and 2β subunits
3. 1

Question 12

1. what is hyperekplexia caused by?
2. what are the effects of these mutations?
3. name 4 symptoms seen in infants
4. name 3 symptoms seen in adults.
5. what is the treatment? What is the action of this drug?

Answer 12

1. mutations in the glycine receptor
2. there is disruption of the balance of excitatory and inhibitory inputs from the CNS. Because glycine receptors normally provide inhibitory inputs, this mutation causes a LOSS OF INHIBITION
3. hypertonia, enhanced startle reflex particularly in response to auditory and tactile stimuli, apnea (increased risk of SIDS). Life threatening condition
4. enhanced startle reflex in response to auditory and tactile stimuli, prone to falls and injuries, occasional uninduced nocturnal convulsive seizures.
5. clonazepam (a benzodiazepine) - activates GABA_A receptors (an alternative inhibitory pathway)

Question 13

1. Name 2 dominant mutations that were studied by expressing the mutant α subunits in xenopus oocytes?
2. what did they find in these mutants (compared to WT) in terms of glycine sensitivity?
3. what did they find in these mutants (compared to WT) in terms of Voltage dependent desensitisation of the channels?
4. What are the conclusions of these findings?

Answer 13

1. L276Q & L250T
2. while WT channels were maximally activated at 500μm of glycine, L276Q channels were maximally activated at 10mM of glycine, at which the size of the current was also significantly smaller
3. WT channels showed Vdep desensitisation at -70mV but not at +70mV. L250T channels showed Vdep desensitisation to glycine at both -70mV and +70mV
4. the reduced max current and sensitivity to glycine means that there is less inhibition, thus excitatory inputs are more likely to produce an AP. Also, channels are more likely to become desensitised to glycine, therefore if they do manage to become activated, they wont stay activated for long.

Question 14

1. what is the main defect in recessive mutants?
2. How was this shown?

Answer 14

1. mutant glycine receptors fail to be trafficked to the surface
2. fluorescence imaging revealed the presence of WT and dominant mutant channels both intracellularly and at the cell surface. recessive mutants were only seen intracellularly.

Question 15

1. how were transgenic mice models created?
2. in terms of the whole cell patch clamp studies in transgenic mice, what were used to ensure that currents measured were only mediated by Glycine, GABA_A and glutamate receptors?
3. How was the glycine component of PSCs isolated?
4. Name 3 phenotypical characteristics of the transgenic mice. What is this phenotype similar to?
5. What was the state of Glycine and GABA_A components of the PSPs in the transgenic mice?

Answer 15

1. expressing either WT GlyR_α1 subunit or the hyperekplexia specific, dominant 217Q GlyRα1 subunit
2. chemical blockers were used
3. kynurenic acid and bicuculine were used to inhibit Glutamate and GABA_A components
4. tremor and disturbed righting; handling induced tremour; hind feet clenching. Similar to spastic and spasmodic mice which carry mutations in the beta or alpha1 subunits of the glycine receptor respectively
5. Gly components was significantly reduced. Even larger reduction on GABA_A component was seen.