辅导 Phar0011: Molecular Pharmacology 2024-2025辅导 C/C++编程

Phar0011: Molecular Pharmacology

2024-2025

Level-6 Students: Sodium channel block summative data analysis exercise.

In this exercise the effect is studied of two blockers of sodium channels, tetrodotoxin and procaine, on the compound action potential recorded extracellularly from an isolated frog sciatic nerve. The aim of the exercise is to test the hypothesis that the two blockers act on the same channel site: this is done by measuring their effect when applied individually and then simultaneously (see Theory appendix).

Tetrodotoxin (TTX) is a powerful paralytic toxin which is found in Tetraodontid fish, including puffer fish, the Japanese fugu.  It has almost no therapeutic use, but is a valuable tool in experimental neurophysiology.

Procaine is a synthetic agent which was used clinically as a local anaesthetic.

The APPENDIX outlines the theory needed to predict the proportion of channels blocked by TTX or procaine or a mixture of the two.

Draw a diagram of a voltage-dependent sodium channel and label the diagram with the main functional domains of the channel.  Indicate on your diagram the regions ofTTX and procaine binding.

METHOD

Frog sciatic nerve was used to make the measurements to be analysed in this exercise. Briefly, the

sciatic nerve was dissected from apithed frog and mounted in a recording chamber, with stimulating electrodes at the distal (knee) end of the nerve and recording electrodes at the proximal end. Typical stimulation parameters are voltage pulses of 0.1 ms duration at a frequency of 0.1 s-1.   The stimulus strength was adjusted to twice the voltage required to give the maximal compound action potential amplitude. The central section of the nerve was cleaned of its connective tissue sheath in order to allow the blockers a faster access to axonal membranes.  This de-sheathed portion of the nerve was in the centre compartment of the nerve bath and application of drug solutions was restricted to this compartment by vaseline seals around the nerve that prevent drugs from reaching the stimulating and recording chambers.

EXPERIMENTAL RESULTS

i) Control action potential. The compound action potential recorded with an extracellular electrode has a biphasic waveform.

Question 1. Why is the compound action potential biphasic?

ii) The action potential conduction velocity was measured to be 28 ms-1.

Question 2. What does this tell you about the types of nerve fibres which contribute to the action potential in frog sciatic nerve?

The height of the upward deflection of the action potential was recorded at one minute intervals.  Once the action potential amplitude was reasonably steady, the experiment was started.

Experiment 1. Inhibition by 125 nM TTX.  The solution in the chamber was changed to include 125 nM TTX. The amplitude of the compound action potential was measured every minute, until it had reached a stable value and the results are shown in Table 1.

Question 3. Plot a graph of action potential amplitude against time for the block with 125 nM TTX.  What factors do you think determine the time course of the block by TTX?

The nerve was then washed thoroughly, and the action potential allowed to recover to the control value.

Experiment 2.  Dose-response curve to TTX.  The aim was to construct an 8-point cumulative dose-inhibition curve for TTX.  Care was taken to measure responses at equilibrium (i.e. when the action potential amplitude  is no longer changing) and that a full recovery to baseline is reached after washout of TTX.

Question 4. Make a plot of the % decrease in action potential amplitude against log(TTX concentration) for the data shown in Table 2.

Question 5. Estimate the IC50  for block of the action potential by TTX.

Question 6. Make a Hill plot of the data and estimate the Hill coefficient (nH).  What do you conclude from the value of the Hill coefficient?

Question 7. Finally, fit these data to the Hill equation using non-linear least-squares fitting in Excel.  How does the value of the Hill coefficient obtained by curve fitting compare to the value you obtained graphically?

Experiment 3.Inhibition by 1.5 mM procaine.   1.5 mM procaine is applied to the nerve and the decrease in the action potential amplitude recorded (the % inhibition is given in Table 2). For the purpose of data analysis, this procaine concentration (1.5 mM) is called P.

Question 8. Response to a procaine-TTXmixture.  From the dose-response curve to TTX, find a concentration of TTX that would give the same %inhibition as the procaine concentration P and call this chosen concentration T.

A solution was made up containing 50% of T plus 50% of P (this was simply achieved by mixing equal volumes of the solutions containing [P] and [T]) and applying to the nerve. The effect of this mixture on the action potential was greater than that produced by [T] alone (the % inhibition is given in Table 2).

Question 9. From the TTX dose-response curve, estimate the concentration of TTX that would give the same inhibition as the 50%/50% mixture and call this concentration T’ .  Accurate matching is important here to allow precise calculations from the data.

CALCULATIONS

The experiment is designed to give equieffective concentrations of procaine and TTX, called P and T, respectively. Since they have equal effect, they must produce equal sodium channel occupancy; hence cP  = cT (from Eq. 4, see Theory appendix).

If the drugs compete, then the mixture containing 0.5P + 0.5Twill produce a block equal to that produced by P or T, (and T’ = T ) i.e. p(mixture)= p(T) = p(P)

If the drugs do not compete, then the mixture will produce a greater block than P or Talone, i.e. T’ > T.  Equation (9) (appendix) should apply.  In this case, use T'to calculate KP  and KT  (the equilibrium constants for the binding of each drug to the sodium channel):

Question 10. What values of Kp  and KT did you estimate from your data?

PROBLEMS TO SOLVE

Question 11.  Would competitive agents still produce the same effect if you mixed equiactive solutions of  them in the proportions f and 1-f, i.e. if you mixed fP + (1 -f)T, where f is any fraction (0 <f < 1), or does it work only for f = 0.5, as used in the experiment?

Question 12. Having obtained KT  from the experimental data using equation (10), calculate the occupancy of the Na+ channel by TTX at the concentrationsofTTX tested in the experiment (use the Hill-Langmuir equation for the calculation).  Plot the results on the same graph as your TTX dose-response curve.

Question 13. What proportion of channels is blocked at a TTX concentration equal to KT  ?  What is the % inhibition of the compound action potential at this TTX concentration?

Question 14. What proportion of channels must be blocked to obtain 50% inhibition of the compound action  potential?  Discuss (e.g. what does a dose-response curve tell you about receptor occupancy and KT  ?  Why?).

Question 15. Combination therapy is one approach to minimising drug side effects.  From your data, estimate what will be the minimum concentrations of procaine and TTX that you could combine in order to achieve a    total of 80% bock of the sodium channels.