As we discussed earlier, the purpose of this study is to see if carbon dioxide and other small molecules like it enter cells by pushing their way through the cell membrane or by way of pores in the membrane that allow their passage. In particular,the authors of this study wanted to know if the protein aquaporin-1 lets CO₂ enter cells. Here's how they went about it:

They started with a Xenopus laevis oocyte (African clawed frog egg cell) and injected it with carbonic anhydrase and cRNA coding for aquaporin-1. After allowing the cell to transcribe the cRNA and make aquaporin-1, the cells were simultaneously impaled with micro-electrodes that monitor the pH of the cell.

As CO₂ enters the cell, carbonic anhydrase changes it to carbonic acid, which lowers the cellular pH. Therefore, more CO₂ entering the cell leads to a lower pH, which is then detected by the electrodes.

Q. What's carbonic anhydrase, and what's it for?

A. When CO₂ is enters the cell, the enzyme carbonic anhydrase catalyzes the following reaction,

and Xenopus laevis oocytes create little if any of it on their own. (See why this is important in the answer to the next question).

Q. How the heck does a decrease in pH signal increased CO₂ permeation?

A. The carbonic acid produced by carbonic anhydrase from CO₂ dissociates by the following reaction,

raising H⁺ concentration in the cell and lowering pH. Therefore, a fall in pH signals an increase in CO₂ uptake.

Q. How does cRNA make aquaporin-1?

A. It doesn't – the cell does! Think of cRNA as a recipe for a protein, in this case aquaporin-1. When injected into the cell, the cell reads this recipe to make aquaporin-1 from various amino acids. The protein created is then transported to the cell membrane, where it resumes normal function.

Q. Why did they use Xenopus laevis oocytes?

A. The Xenopus laevis oocyte had several features that made it the perfect cell for the job.

First, the Xenopus laevis oocyte has very low baseline permeability. This makes it easier to detect raised levels of CO₂ uptake in the cell because it would lead to a much larger change in pH.

Second, the Xenopus laevis oocyte is fairly large. The microelectrodes measure pH changes, but it would be incredibly difficult to measure these changes in a small cell because they would progress far too rapidly for the electrodes to detect. The intercellular pH changes at a much slower pace in larger cells, making the drop in pH noticeable to the microelectrodes.

Another important feature of the Xenopus laevis oocyte is its responsiveness to foreign RNA. While many cells do not process foreign cRNA, this cell does so readily. The oocyte's tendency to transcribe foreign cRNA is very important, since not doing so would preclude the aquaporin-1 pores from being created and expressed on the cell membrane.

Also important is the Xenopus laevis oocyte's resistance to H⁺ from its surroundings. Because the oocyte's membrane appears to allow only minimal passage of H⁺ into the cell from outside, we know that any pH changes inside the cell are from CO₂'s entrance, not the entrance of H⁺.

Finally, the Xenopus laevis oocyte is tough. Many cells would not survive the multiple injections and long-lasting impalements that this oocyte endures. If the cell chosen were to die or bear large breaks in the membrane, the experiment would fail.