Your Assignment: The big question is: What type of ion channels are the MACs in

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Your Assignment: The big question is: What type of ion channels are the MACs in our neuron? They are mechanically?gated, but are they only permeable for a particular ion species or are they unselectively permeable for all kinds of anions and/or cations? If they are permeable for one specific ion species which one listed in Table 1 is the most likely candidate?  The results of our experiments shown in Fig. 1 allow us to come up with a solid hypothesis what kind of ion specific MACs we have to deal with.  Analyzing the current behavior at the different membrane potentials you will find the clues to solve this problem! Based on your findings and observations you will have to: I) Formulate a hypothesis describing (and explaining why you come to this conclusion) the Type of Ion Channel our MACs are, i.e. for what ion species they are permeable. (8 pts)

II) Describe a strategy for a follow?up experiment that you could do to verify your hypothesis, i.e. confirm what ion species is able to flow through these MACs.  (6 pts).  

Scenario: For our project we use cultured mechanosensory neurons. These neurons are well established model cells with intracellular and extracellular ionic environments known (listed in Table 1). Previously you calculated important parameters like equilibrium potentials (E) for Na, K", Ca2+, Mg2+, and Cl, and the membrane resting potential (VM) (Table 2; use the listed numbers!). All experiments are carried out at 30 'C. Goal of you study is to determine the nature of mechanically activated ion channels (MACs) fond in these neurons. The drugs TTX (to block voltage-gated Na*-channels) and TEA (to block voltage-gated K'-channels) has been applied to prevent voltage-gated channels to open and influence the observed MAC-currents. Extracellular Concentration [XlouT 240 mM 8.5 mM 5 mM 6 mM 580 mM Intracellular Concentration Membrane Permeability P in resting neuron lon IN 37 mM 340 mM 0.02 mM 0.08 mM S5 mM 10 100 Not significant Not significant Cl Table 1: Intracellular and extracellular ion concentrations, and membrane ion permeabilities of the resting cell membrane of mechanosensory neurons (from Assignment #1), we can assume that ion concentration do not change during the experiments. No+ Kt Eca2 EM +0.0488 v 0.0963 V 0.0721 V +0.0564 v 0.0622 V 0.0616 V +48.8 mV 96.3 mV +72.1 mV +56.4 mV 62.2 mV 61.6 mV Table 2: Equilibrium potentials and membrane resting potential of mechanosensory neurons. Use the numbers listed in Table 2, not the ones you calculated in the first assignment. They may be the same or similar, but we want to make sure everyone uses the same starting point.

Explanation / Answer

Hypothesis: As the plot in Fig. 1 indicates zero current at Vm = +70 mV, which from table 2 is more closer to that of Ca2+ ions

So, this indicates the ion channels studied are Ca2+permeable ion channels.

Justification: Now, if we go into calculations, The current Im is proportional to difference of Vm (applied) and Vequi of concerned ion

So, whenever Vm (applied)-Vequi comes out to be positive for a cation, it means the current (Im) is positive and there is net outflow of the cation

whenever Vm (applied)-Vequi comes out to be negative for a cation, it means the current (Im) is negative and there is net inflow of the cation

Now, if the cation under consideration is K+, at zero Vm (applied), Im would be [0-(-96.3)] i.e. +ve value which is not visible from the plot. Same holds good for Cl-. Considering Na+ and Ca2+, at Vm (applied) = +60 mV, Im for Na+ comes out to be [+60-(+48.8)] i.e. a positive value which is not in present case. Same thing happens for Mg+2.

Im for Ca2+ comes out to be [+60-(+72.1)] i.e. a negative value which is the case in the plot. At all the values of applied Vm above +70 mV there is +ve Im indicating outflow of Ca2+ ions while applied Vm lower than +70 mV gives -ve Im indicating inflow.

Follow up strategy:

The hypothesis can be experimentally tested by creating very low (less than physiological range) concentrations of Ca+2 in the extracellular fluid of test neurons in vitro. This will change the Vequi of Ca2+ and hence the results will confirm by modifying the plot.

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