You are studying the ability of your cultured cells to increase Ca == intracellu

Question

You are studying the ability of your cultured cells to increase Ca==intracellularly in response to a newly identified agent you have extracted from a soil fungus during a pharmaceutical screen. You are trying to identify HOW that increase in [ Ca==] is occurring, ie via entry of Ca from extracellular medium OR the ability of this novel agent to elevate intracellular [ Ca==] via activation of IP3 production. Outline THREE DIFFERENT approaches you would use, and the outcomes you would expect IF the increase in intracellular [ Ca==] was from extracellular sources or release of Ca== from intracellular stores.

Use the format below to describe your experimental strategy, and then the expected outcomes ( i.e., IF…..THEN…. )

Experimental Strategy

IF Ca++ from EXTRACELLULAR STORES

If Ca++ from INTRACELLULAR IP3-responsive stores

1)

2)

3)

Experimental Strategy

IF Ca++ from EXTRACELLULAR STORES

If Ca++ from INTRACELLULAR IP3-responsive stores

1)

2)

3)

Explanation / Answer

1. The experimental strategy would be using a growth medium which is devoid of Ca ions. This can be done by adding Ca chelators to the medium. Then add the newly identified to the cells and measure the increase in Ca ions.

If the Ca entry is from the extracellular environment, then the new agent cannot show its activity of increasing Ca ions as the medium is devoid of Ca ions.

If the Ca release is from IP3 activation, then you can see the increase in the Ca levels. As the chelation of Ca in media won't affect the Ca release intracellularly.

2. The second strategy would be to use an IP3 inhibitor, which specifically blocks the IP3 signalling and release of Ca ions from intracellular stores.

If the Ca is entering from extracellular source, there would no effect on Ca increase by usage of IP3 inhibitor.

If the Ca is from the intracellular sources, then IP3 inhibition would not cause Ca increase in the cell after the addition of the new agent.

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