MEDSCI 309 Biophysics of Nerve and Muscle SEMESTER TWO 2019
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MEDSCI 309
SEMESTER TWO 2019
MEDICAL SCIENCE
Biophysics of Nerve and Muscle
SECTION A (100 marks)
Answer ANY FOUR of the following FIVE questions (each in a separate script book).
Show all working and use SI units throughout.
1. Consider a cell with membrane approximated electrically as follows:
The cell is assumed to have the following ionic and electrical properties: [K]intra = 150 mmol/L [K]extra = 5 mmol/L, [Na]intra = 10 mmol/L, [Na]extra = 140 mmol/L,
gK = 150 nS, gNa = 10 nS, F = 96500 C/mol, R = 8.31 J/K/mol, T = 300 K, Cm = 0.01F/mm2 (specific capacitance).
(a) (4 marks) For the given parameters, use the Nernst Equation to calculate the reversal potential for sodium ions, ENa, to the nearest mV (hint: remember to include the ion charge number).
(b) (4 marks) For the given parameters, calculate the reversal potential for potassium ions, EK, to the nearest mV.
(c) (5 marks) What is the membrane potential, Vm, of the cell, to the nearest mV?
(d) (6 marks) At this membrane potential, Vm, what is the magnitude of the current flowing through the sodium and potassium channels? Give your answer to the nearest 1/10 of a A. Briefly comment on your answers.
(e) (6 marks) What current, Ic, would be needed to clamp the membrane potential at - 10 mV? Assume that there are no voltage-dependent changes of parameters. Give your answer to the nearest 1/10 of a A.
2. The figure below shows force responses from a “skinned” skeletal muscle fibre subjected to repeated ‘load–release’ cycles in which the sarcoplasmic reticulum (SR) was loaded with Ca2+ for the indicated time and then the SR Ca2+ released by exposing the fibre to ‘full release solution’ .
The first response in the series was that elicited upon releasing the endogenous SR Ca2+ (‘Endo’). The final response shows the maximum force elicited by directly activating the contractile apparatus in a heavily Ca2+ ‐buffered solution.
The ‘load solution’ contained pCa 6.7 with 1, 3 or 10 mM cytoplasmic free Mg2+ The ‘full release solution’ contained 30 mM caffeine, 0.05 mM free Mg2+ (total Mg2+ of 2.1 mM) and 0.5 mM free EGTA (pCa 8.5)
Time scale: 10 s during SR Ca2+ release; 30 s for maximum Ca2+ ‐activated force.
J Physiol. 2016 Jan 15; 594(2): 469–481. (Figure 1A.)
Answer the following with respect to the above study:
(a) (3 marks) Using the units displayed on the vertical axes, identify the three measures being plotted in A, B and C, and describe them in terms of the nerve axon.
(b) (2 marks) What advantage does a skinned muscle fibre preparation have? (c) (2 marks) What is ‘pCa’?
(d) (1 marks) What do the vertical arrows represent?
(e) (1 marks) What do the horizontal bars represent?
(f) (2 marks) What mechanism was used for activation of force development in this study?
(g) (2 marks) What is the approximate duration of muscle fibre contraction in
response to the release solution?
(h) (1 marks) How do you think force was measured?
(i) (2 marks) Why does force increase and decrease with each intervention?
(j) (2 marks) Why do you think traces 8, 9 and 10 (numbering from the left) are all of similar size?
(k) (2 marks) Why is the force greatest in the final trace?
(l) (5 marks) What do you conclude about the role of Mg2+ from these experimental data? Explain your answer with reference to the data shown.
3. These data show the effect of phospholamban knockout (deletion) on mouse cardiomyocyte Ca2+ handling and response to β-adrenergic stimulation via isoprenaline. From Briston et al, 2014 Cardiovascular Research.
Abbreviations: Phospholamban (PLN), wildtype ‘control’ mouse (WT), phospholamban knockout mouse (PLN-KO), caffeine (caff), isoprenaline (Iso), intracellular Ca2+ concentration ([Ca]i), Na+-Ca2+ exchanger current (INCX), rate constant of Ca2+ transient decay (kSYS), sarcoplasmic reticulum Ca2+ content ([SR]).
(a) (5 marks) Explain the effect of caffeine on the Ca2+ transient
(b) (5 marks) Explain the effect of isoprenaline on the Ca2+ transient
(c) (10 marks) Explain the effect of phospholamban knockout on cardiomyocyte Ca2+ handling, with specific reference to figure panels and comment on the
mechanisms involved
(d) (5 marks) Why does isoprenaline have less of an effect on the cardiomyocyte Ca2+
transient in the phospholamban knockout mice relative to the wildtype control mice?
4. The figure below is from the paper “The effect of internal and external potassium concentration on the membrane potential of frog muscle” Adrian R.H. (1956). In these experiments, resting membrane potential was measured from Sartorius muscle in various external solutions. Specifically, the external solution was modified by substituting equimolar concentrations of KCl and NaCl to obtain solutions with a range of [K+]O . ‘Sulfate solutions’ were made using the membrane impermeable sulphate ion as the anion (e.g. K-sulphate instead of K-chloride). NOTE that the sign of the values on the ordinate are opposite to those of the present-day convention, reflecting the fact that resting membrane potential was calculated as Vo – Vi.
(a) (5 marks) At what temperature (ºC) were these experiments conducted at?
Remembering that 2.3log(x) = ln(x)
(b) (3 marks) From the figure above, what evidence is there that the Nernst equation
is insufficient for describing the resting membrane potential?
(c) (2 marks) What effect does extracellular Na+ have on the measured membrane
potential?
(d) (10 marks) Compare the measured values for resting membrane potential in the
chloride and sulphate solutions.
a. (6 marks) By comparing the measured values of resting membrane potential between the chloride and sulphate solutions in the overlapping range of [K+]o (10, 25 and 50 mM), what can you infer about the role of Cl- in determining resting membrane potential?
b. (4 marks) Comment on the range of values for [K+]o used with the sulphate solution and why this solution was used with that range. Remember that sulphate is a membrane impermeable ion
(e) (5 marks) What key value can be determined from an intercept of the bottom plot?
5. The accompanying Figure (modified from Funk et al, Respiration Physiology 110: 125- 137, 1997) shows response of hypoglossal (XII) motoneurons (Im) to application of Phenylephrine (PE; 10 M). Panel C shows XII motoneurons with inspiratory related rhythmic activity in response to PE (10 M) and panel D show individual inspiratory synaptic current under control and in the presence PE (10 M) Panel E shows XII motoneuron in response to a 10 mV pulse in control and in the presence PE (10 M).
(a) (2 marks) From panel C estimate the amplitude of the PE-induced current.
(b) (2 marks) From panel D estimate the change in inspiratory synaptic current in
response to PE
(c) (3 marks) What is the input resistance of the cell under control condition?
(d) (3 marks) What is the input resistance of the cell under PE condition?
(e) (2 marks) Based on the change in input resistance, has the excitability of the
motoneuron increased or decreased?
(f) (10 marks) Based on the change in input resistance and excitability, propose a
mechanism by which PE impacts neuronal activity.
(g) (3 marks) If you were to apply tetrodotoxin (TTX) to the preparation, what would
you expect to happen to the inspiratory drive and the PE-induced current?
SECTION B (80 marks)
Answer ANY TWO of the following FIVE questions (each in a separate script book).
Feel free to employ sketches and figures if appropriate, but ensure that they are clear, well- labelled and relevant to your discussion.
6. Write a structured essay to succinctly address the following questions in the context of action potentials in nerve axons.
(i) What is biophysics and why might scientists be interested in the biophysics of electrical activity in nerves?
(ii) What does a voltage clamp technique do and how does it work?
(iii) Prior to the development of this technique in the late 1940s, what measurements
could be made on nerve axons?
(iv) What is the simplest way to determine Na+ and K+ ionic membrane currents in a
voltage clamp experiment?
(v) How are the ion channel conductivities determined?
(vi) Using the potassium ion channel model of Hodgkin and Huxley as an example,
describe how ion channel conductivities can be used to find the channel rate parameters as functions of the clamp potential V? (Recall that for this example: gK = K n4 , n = n ∞ − (n∞ − n0 )e−t⁄Tn (for constant V), an = n ∞ ⁄Tn and Fn = (1 − n∞ )⁄Tn .)
(vii) What is something surprising you learned from the Action Potentials and
Conduction section of this course and why do you think it is important to know?
7. Write an essay on the different ways that force can be modulated in cardiomyocytes, focusing on the underlying excitation-contraction coupling mechanisms. Include specific mention of:
(a) Intrinsic cellular mechanisms (i.e. properties of the cardiomyocyte) (b) External stimuli e.g neurotransmitters, pharmacological drugs
8. Early experimenters thought that postsynaptic excitation of skeletal muscle released some substance from the sarcolemma that diffused to the centre of the cell, causing contraction.
Write an essay in which you
(i) Provide a detailed description of skeletal muscle excitation-contraction coupling
(ii) Describe the experiments that ruled out diffusion of some activating substance
(iii) Outline the experimental evidence that supports our current understanding
9. The 3 equations below can be used to mathematically model the resting membrane potential to varying success. Describe the basis for each equation (1 to 3), including the advantages and/or disadvantages of each to model resting membrane potential. Start your essay with an explanation of the biophysical mechanisms that lead to the establishment of the resting membrane potential.
where
1
2
3
10. In skeletal muscle, the most unambiguous estimate of the magnitude of activation heat has been made when preparations have been stretched to the point of zero overlap of the thick and thin filaments. In cardiac muscle, this technique is not successful since it is practically impossible to stretch cardiac sarcomeres beyond about 2.7 µm without causing irreversible damage.
The above statement is paraphrased from Gibbs et al. (1988) “Activation heat in rabbit cardiac muscle” .
Write an essay in which you explain:
(i) How you would estimate activation heat in cardiac muscle.
[In your answer, include a plot of total heat as a function of isometric force to illustrate whether activation heat is length dependent or length independent]
(ii) The cellular and ionic processes that liberate activation heat.
[Bonus marks will be given to answers that detail the percentage contribution of each of the processes to activation heat]
(iii) The effect of increased activation heat on cardiac efficiency.
[In your answer, describe an experimental method you would use to increase activation heat, and its effects on external work output, cross-bridge heat and mechanical efficiency]
2022-10-31