Reference no: EM133035090

PSYC 2260 Fundamentals of Neuroscience - Mount Saint Vincent University

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 IR) that we can measure.

As a precaution we applied drugs like TTX and TEA to prevent voltage-gated Na+-channels and voltage-gated K+-channels from influencing the observed membrane currents during our experiments, i.e., we are sure that we measure only membrane currents carried by ions crossing the membrane through MACs we control!

Use the Equilibrium Potentials listed in Table 1, not the ones you calculated in the first assignment. Ideally the numbers you calculated should be close or the same, but by using the ones in Table 1 we make sure everyone works from the same starting point.

Experiment:
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 IR 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.

Your Assignment:

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
I) 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, and

II) 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 II):

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)?

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 ions) affect the membrane potential VM under non-voltage-clamp conditions? Will VM change or not, and if it changes will VM become more negative (hyperpolarize) or less negative (depolarize)? Explain your answer!

V) 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 I) and follow-up experiment II)?

Sounds though? Think about what you learned in class and use it to interpret information can be read out of Figure 1 and Table 2. Ask yourself the following questions before writing up your assignment. Think before you write, and proofread not only for spelling and grammar, but also for logical flow in your argumentation.
• What forces drive the movement of ions across the cell membrane through open ion channels (here MACs) and determine the direction of ion movement at different VM?
• What ion species (Na+, K+, Ca2+, Mg2+, or Cl-) can carry the observed current across the cell membrane at the given VM, i.e., move in the required direction across the membrane at the different VMs to result in the observed current IR?
• Why did we care about calculating and knowing equilibrium potentials prior to our experiments?
• How can we prevent ions from flowing through open ion channels or speed-up their flow in experiments like this one?
• What will happen if we change the extracellular concentration of one ion species? Will this change the resulting membrane current carried by this ion species and the resulting data plot?

Attachment:- Fundamentals of Neuroscience.rar