Scenario: For your research project you use neurons that are kept in cell culture. These neurons are well established model cells with known intracellular and extracellular ionic environments (Table 1). Parameters like equilibrium potentials and membrane resting potential have been calculated (see Table 1). All experiments are carried out at 20 C. Goal of your study is to determine the nature of mechanically gated ion channels (MACs) expressed by these neurons. A 200 ms long mechanical pressure stimulus is used to open the MACs and the channels remain in open state for the duration of the stimulus, allowing ions to cross the membrane and carry a mechanically induced membrane current (receptor current I) that we can measure. As a precaution we applied drugs like TTX and TEA to prevent voltage-gated Na-channels and voltage-gated ki-channels from influencing the observed membrane currents during our experiments, l.e., we are sure that we measure only membrane currents carried by Ions crossing the membrane through MACs we control! I Membrane Resting Potential VM @ 20 C lon Species Extracellular Intracellular Equilibrium Concentration Concentration Potential [X]out [X]IN Ex @ 20 C Na 260 mm 35 mm + 50.7 mv K 8 mm 340 MM 94.7 mV Cal 20 mm 0.1 mm + 66.9 mV Mg. 60 mm 0.2 mm + 72.0 mV CI 560 mm 60 mm 56.4 mv Vw - 58.4 mv Table 1: Intracellular and extracellular ion concentrations and resulting equilibrium potentials and membrane resting potential at 15 "C. When a mechanical stimulus is applied MACs in the membrane of the neurons open. We measure the resulting ion-carried membrane current (IR), the receptor current, flowing through open MACs across the membrane of the neuron at different membrane potentials, using a recording technique known as voltage-clamping. This method allows use to set and control Vm of the neuron at potentials ranging from -100 mV to + 100 mV (holding potential VH = Vm), and to prevent VM from changing despite a current flowing across the membrane. We can "clamp" and hold the cell at any desired Vm during our experiment to measure the mechanically induced membrane current le at the different "clamped" membrane potentials (see figure 1 below and table 2 in the appendix). Application of TTX and TEA is just an additional "security blanket that is not mandatory for our experiment to work 100 Membrane Voltage V., (mv) 100 50 100 100 300 Membrane Current IR (PA) Fig. 1: IV plot of mechanically induced membrane ion current Ir at membrane potentials VM ranging from - 100 mV to + 100 mV. (see Table 2 in appendix for raw data). Figure 1 shows the result of our experiment as IV-Plot (current vs voltage) with the measured mechanically induced membrane current is measured at different clamped membrane potentials Vi between - 100 mV and + 100 mV. Note: By definition, cation carried inward currents (cations entering the cell) are measured and shown as negative currents and cation carried outward currents (cations leaving the cell) are measured and shown as positive currents. The big question is: What type of ion channels are the MACs in our neurons? Are they selective and permeable for only one ion species, or are they unselectively permeable for several different cation and/or anion species? If they are indeed selectively permeable for only one specific ion species which one is the most likely candidate? How can you tell this by analyzing the provided information (data from our experiments)? Your job is to 1) formulate a hypothesis describing what type of ion channel we can expect our MACs to be, and reasonably explain why and how you come to this conclusion (10 points), and 11) think about, describe, explain, and discuss a possible strategy for a follow-up experiment that we could do to verify your hypothesis (4 points). For part II keep in mind what factors determine an ion's equilibrium potentials and how we could use this to test your hypothesis. In addition, answer the following questions that may help to complete I) and 11): III) Looking at the results (Figure 1, Table 2) describe what forces drive the ion flow across the membrane through open MACs at - 100 mV, -50 mV, 0 mV, +50 mV, and +100 mV. How does changes in Vm determine the direction of ion flow across the membrane, i.e., the membrane current we measured (see Figure 1 and Table 2)? (4 points) IV) Imagine doing this experiment without voltage-clamp, i.e., have the cell at its natural membrane resting potential of VM = -55.7 mV when starting stimulation and ions crossing the membrane through open MACs carrying a membrane current. How would the flow of ions across the membrane (and the current carried by those lons) affect the membrane potential V under non-voltage-clamp conditions? Will VM change or not, and if it changes will VM become more negative (hyperpolarize) less negative (depolarize)? Explain your answer! (4 points) or Vj Will the results of this experiments change if it is conducted at 10 "C? What parameters will change, how does this affect the observed current, and would the change require you to review your hypothesis under 1) and follow-up experiment II)? (3 points) Appendix: 100 80 -60 .40 -20 0 + 20 +40 +60 +80 + 100 V (mv) le (PA) - 530-350 - 250 - 175 - 110 70 . 40 - 25 - 8 + 5 + 8 Table 2: Row Dota for the experiment shown in figure 1