BA, BSc, and MSc Degree Examinations

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1 Examination Candidate Number: Desk Number: BA, BSc, and MSc Degree Examinations Department : BIOLOGY Title of Exam: Membrane transport Time Allowed: 2 hours Marking Scheme: Total marks available for this paper: 100 Sec on A: Short Answer / Problem / Experimental Design ques ons (50 marks) Sec on B: Essay ques on (marked out of 100, weighted 50 marks) The marks available for each ques on are indicated on the paper Instructions: Sec on A: Answer all ques ons in the spaces provided on the examina on paper Sec on B: Answer either ques on A or ques on B. Write your answer on the separate paper provided and a ach it to the back of the ques on paper using the treasury tag provided. Materials supplied: For marker use only: CALCULATOR For office use only: Module total as % DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR page 1 of 10

2 SECTION A: Short Answer / Problem / Experimental Design questions Answer all questions in the spaces provided Mark total for this section: Using an ion selective electrode you measure 0.1 mm Mg in the plasma of a patient and around 1 mm Mg inside red blood cells. You know in a healthy patient the Mg Nernst potential 'E Mg ' (or equilibrium potential) should be ~-60 mv. To what concentration does the red blood cell Mg concentration need to change to reach ~-60 mv? E=RT/zF ln [Mg 2+ ] out /[Mg 2+ ] in (2 marks) The current 10-fold concentration gradient would generate E Mg of ~-30 mv (1 mark). To reach a value of -60 mv, would require a 100 fold gradient (1 mark) so the cellular [Mg2+] would have to be raised from 1 to 10 mm (1 mark). Most students did not get this and got into v complicated calculations when there was no need for it: If z is 1, the equation is E=-60 log [Mg 2+ ] out /[Mg 2+ ] in (recurrent theme in the lectures). With Mg, z is 2 so E=-30 log [Mg 2+ ] out /[Mg 2+ ] in Thus, with 0.1 and 1 mm, E=-30 and with 0.1 and 10 mm E= Explain why the catalytic mechanism of the Lac permease and other carriers is referred to as the 'rocker switch' model and describe its salient features. (4 marks) The rocker switch model refers to conformational changes that consist of tilting alpha helices during the catalytic cycle of many carriers (1 mark). This not only ensures that substrate binding sites are exposed alternatingly to each side of the membrane ('alternating access') (1 mark) but it typically induces changes in binding affinity (1 mark) which is high on the side where substrate is dilute and low where substrate is concentrated (1 mark). Most students got most of this right 3. You are heterologously expressing KCNQ4 in HEK cells. A whole cell recording shows only a small outward current and you suspect you may be working with a defective KCNQ4 protein. Describe the patch clamp experiments you would run to ensure that your current is generated by KCNQ4. If you find that this is the case, how would you determine whether the protein is a wildtype, a gating mutant or a pore mutant. (7 marks) Since you use a heterologous system, the current may not be from KCNQ4 but endogenous channels. (1 mark) To establish the current is mediated by KCNQ4, you need to record from both expressing and control (empty vector or alike) HEK cells and quantitatively compare currents (this should be done in page 2 of 10

3 whole cell configuration, especially if there are likely to be endogenous channels with similar single channel conductance to that of KCNQ4). (2 marks) To distinguish between gating and pore mutants, single channel recordings need to be made to construct I/V plots of both wildtype and putative mutant and single channel conductances should be compared. (2 marks) Currents may simply be low because expression levels are low but in case of a gating mutant, the open time will be altered in the putative mutant which can be established by running long single channel recordings and analysing them for total open and closed time. (2 marks) This required thinking about proper control experiments and application of the patch clamp and was answered poorly though some students got almost full marks 4. A patient presents with long QT syndrome. Discuss how this affliction is caused and whether treatment with nicorandil would be a viable treatment. (4 marks) Long QT syndrome is a heart arhythmia due to reduced activity of V-dependent K channels. This slows down repolarisation because it prolongs time between Q peak (which is ventricle depolarisation that causes the main pumping action) and T peak (which signifies ventricle repolarisation). (2 marks) To some extent this can be treated with K channel openers such as nicorandil (1 mark) but the non-specificity of such medicines often leads to side effects 1 mark). Quite a few students did not know what nicorandil does but almost everyone had the first bit right (mentioning Nav channels as underlying cause was also credited) 5. a) In what organ/tissue does uric acid transport occur and what are the main diseases that result from faulty uric acid transport. (3 marks) Uric acid transport mainly occurs in the proximal tubule of the kidney (1 mark). Its disruption can cause gout(arthritis)/hypertension/kidney stones/kidney failure/bladder stones (2 marks). Almost universally full marks were obtained here b) Describe the role of URAT1 in gout and discuss a research strategy based on a GWAS (genome wide association study), to identify polymorphisms in this transporter URAT1 and how you would use this to devise a treatment. (7 marks) URAT1 is involved in uric acid reabsorption from the urine (1 mark). Overactivity of URAT therefore leads to uric acid accumulation in the blood serum and increased incidence of gout ( 1mark). A GWAS could be employed to screen a large population of individuals for the incidence of gout or high levels of serum uric acid (or other easily scorable traits) (1 mark). If the population is genotyped, an association analysis should reveal how different haplotypes page 3 of 10

4 correlate to disease incidence. (If not genotyped, sequencing of the URAT1 alleles in the population can be carried out). (1 mark). The association study could pinpoint specific polymorphisms (e.g. SNPs) in the coding region focussing on non-synonymous SNPs (involved in transport/selectivity/structure function) (1 mark) whose function can be assessed using bioinformatics/structural data (if the SNPs are in non-coding regions, they could still impact on expression and regulation) (1 mark). Since these findings will give you a handle on parts of the protein that are related to protein function/activity you may be able to devise/modify existing inhibitors (such as the uricosurics: probenecid, benzbromarone, sulfinpyrazone, and losartan) (1 mark). Some very good answers but in many cases only some correct elements were mentioned though many at least identified the principle of associating phenotypes with genotypes. 6. Single-molecule tracking of a cystic fibrosis transmembrane conductance regulator (CFTR) channel labelled with an extracellular tag yielded mean-square displacement (MSD) data which was dependent on its specific location in the plasma membrane. a) Explain how the above MSD data can be used to quantify the diffusive properties of the CFTR channel in membrane regions 1 and 2, and then compare the diffusive properties observed in each region. (4 marks) The initial gradient of the MSD data (1 mark) for the two regions can be analysed to obtain the lateral diffusion coefficient (1 mark). Diffusion in region 1 is much faster than the observed diffusion in region 2 (2 marks). FEEDBACK : Most students were able to explain how MSD data can be used to quantify the 2D diffusive properties of the CFTR channel. However, most page 4 of 10

5 students focused on whether 2D diffusion was restricted or unrestricted. This is the answer to part b of the question. Most students did not compare the diffusive properties in each region, i.e. which channel is diffusing most rapidly in the membrane as determined from the initial gradient of the MSD vs time data? b) Describe how these MSD data sets inform about the long-range mobility of the CFTR channel in membrane regions 1 and 2 of this cell type. (3 marks) MSD data for CFTR channel in region 1 increases in a linear manner (0.5 mark), which is indicative of non-confined two-dimensional diffusion (1 mark). MSD data for CFTR channel in region 2 increases more slowly and quickly reaches a plateau value (0.5 mark), which is indicative of confined two-dimensional diffusion (1 mark). FEEDBACK : Many students answered this question completely, though they typically did it in the answer to part a. c) What determines the time-resolution in these MSD experiments? (1 mark) Time-resolution is determined by the frame-rate at which the video microscopy data is acquired. FEEDBACK : Most students answered this question correctly. d) What are the benefits of using an extracellular tag in these single-particle tracking experiments relative to a GFP fusion to the CFTR channel? (2 marks) Extracellular tag preserves interactions between the cytoplasmic domains of CFTR and intracellular proteins (1 mark) and avoids background fluorescence from intracellular GFP fusion protein (1 mark). FEEDBACK : Most students answered this question completely. e) Adding 10 histidine residues to the C-terminus of the CFTR channel increased the long-range mobility of the channel in the plasma membrane. How might the observed MSD data for region 2 be explained based on these results? (4 marks) page 5 of 10

6 The 10xHis residues at the C-terminus would mask the PDZ binding motif in the cytoplasmic portion of the CFTR channel (1 mark), which in turn would prevent interaction with NHERF1 and the cytoskeleton (1 mark). This implies that mobility in region 1 is CFTR channel which is not anchored (1 mark), and mobility in region 2 represents channel anchored to the cytoskeleton via interactions with PDZ domain containing proteins (1 mark). FEEDBACK : Many students understood that an intracellular molecular interaction mediated by the C-terminus of the CFTR channel was causing restricted 2D diffusion, and that the 10xHis tag masked the C-terminus. Marks were typically lost for a lack of correct detail about how this might be explained. page 6 of 10

7 7. Below is the Na + current-voltage relationship of a hippocampal pyramidal neuron from a mouse measured using whole-cell patch clamp recording. The internal solution contained 150 mm KCl and 5 mm NaCl. The external solution contained 5 mm KCl and 150 mm NaCl. The external solution also contained 30 mm TEA-Cl and 0.3 mm CdCl 2 to block K + and Ca 2+ currents, respectively. The whole-cell capacitance was measured to be 40 pf. a) What is the peak current density? (1 mark) -10 pa/pf (1 mark) b) What is the reversal potential? (1 mark) +80 mv (1 mark) c) Why is the current non-linear between -50 mv and -10 mv? (2 marks) The Na + channels are voltage-gated (1 mark). Some channels start to open at -50 mv, but not all channels are open until around -10 mv (1 mark). d) Explain what would happen to the reversal potential if TEA-Cl was removed from the external solution. (2 marks) Removal of TEA-Cl would permit K + channels to open, allowing K + efflux (1 mark). The reversal potential would shift to a more negative value because it would be dependent on both Na + and K + (1 mark). page 7 of 10

8 e) Design an experiment you could perform to test the involvement of β1 subunits in regulating this Na + current and describe what you would expect to find. (3 marks) Silence β1 expression in cultured neurons using RNAi, and/or use neurons from Scn1b null mice (1 mark). Quantitatively compare Na + current properties (size, gating, kinetics, etc.) recorded from these β1-deficient neurons with Na + currents recorded from wildtype control neurons (1 mark). Loss of β1 would be expected to reduce size of Na + currents and may shift activation/inactivation to more depolarised potentials (1 mark for either). page 8 of 10

9 SECTION B: Essay question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B Mark total for this section: 50 EITHER A) Design and discuss an experimental approach that would allow you to determine the Michaelis-Menten parameters of an acetylcholine accumulating transporter, located in synaptic vesicles, and how the transporter is energised. You would start by generating some sort of experimental system that allows you to look at the transporter without interference from other transporters. For example, by isolating the synaptic vesicles (if expression is high) and using radiolabelled acetylcholine. Alternatively, the protein can be purified and reconstituted in artificial liposomes that do not contain any other transporters. To establish the Km and Vmax, quantification of acetylcholine fluxes, as a function of added substrate, can be carried out, for example using radiolabelled acetylcholine. (Care has to be taken that unidirectional flux is measures, so preliminary epx may be necessary to establish linearity of uptake during the uptake assay). To study the energising mechanisms, flux magnitude can be measured in the presence/absence of driver ions (in the form of gradients of H+ and Na+), e.g. by manipulating the external ph and/or [Na+]. If the acetyl choline flux is sensitive to the ambient Na+ gradient or H+ gradient it constitutes good (though certainly for ph, not conclusive) evidence for coupling. To test for membrane potential driven transport, you can apply different electrical gradients (using different K+ concentrations and valinomycin to clamp the membrane potential) and test for their effect on acetylcholine flux. An electrophysiological approach (e.g. voltage or patch clamp) could be appropriate (in whole cell mode) but only if transport is electrogenic. Since the transporter function is to accumulate acetylcholine, an antiport (typically electroneutral) is the most likely mechanism. OR B) Using specific examples, discuss how preclinical in vitro and i n vivo page 9 of 10

10 studies have contributed to our understanding of the role of Nav1.1 channels in Dravet syndrome. A model answer should start with a definition of Dravet syndrome, indicating its location on the severe end of the spectrum of heritable epileptogenic disorders, relating this to the functional effect of mutations in the Na v 1.1 gene. The essay should describe in vitro studies characterising the effect of epilepsy-causing mutations in Na v 1.1 on size/activation/inactivation kinetics of Na + currents in heterologous cells using patch clamp recording. The essay should then describe experiments performed using mouse models of Dravet syndrome, in particular the effect of global Na v 1.1 deletion/haploinsufficiency on behaviour in mice (survival, righting reflexes, feeding, EEG recordings of seizure activity), followed by effect on Na + current in bipolar vs. pyramidal neurons, and relate these to changes in electrical excitability and the neuronal and network level. The essay may also refer to more recent studies using targeted deletion of Na v 1.1 in specific neuronal subpopulations. The essay should relate these findings to the prevailing model: Na v 1.1 is predominantly expressed in bipolar neurons, which have an inhibitory effect on network activity. Therefore, loss of function in these neurons may result in increased network excitability in the brain, including Dravet Syndrome. page 10 of 10