My Hypothesis: The ion types permeable for one specific ion species in Table 1 i

Question

My Hypothesis: The ion types permeable for one specific ion species in Table 1 is K+.

Question: 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. ?

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', Mg, and CI, 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 bloclk 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. lon Extracellular Concentration Intracellular Concentration Membrane Permeability P in resting neuron Na K+ 240 mM 8.5 mM 5 mM 6 mM 580 mM 37 mM 340 mM 0.02 mM 0.08 mM 55 mM 10 100 Not significant Not significant 24 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 concen tration do not change during the experiments. ENG+ +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 Eca2* VM Table 2: Equilibrium potentials and membrane resting potential of mechanosensory neurons.

Explanation / Answer

CA protein production and purification Stratergy.

In Eschericia coli cells cloned plasmids can be used for expression of
CA proteins.Ion Exchange Chromatography -IEC, Immobilised Metal Affinity Chromatography
IMAc and Size Exclusion Chromatography (SEC) can be used to purify
these proteins.

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