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Potassium Channels - Determining the functional amino acids!

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Potassium Channels - Determining the functional amino acids!

Postby dharavsolanki » Tue Oct 05, 2010 4:44 am

Hi!

I am working on a report right now.

It was assigned to us when we were discussing the selective potassium channels in cells which didn't allow the passage of sodium ions through them.

We had to determine the amino acids that are actually functional in the channel. One general logical method described to us was to use site directed/point mutagenesis. Using this, we had to change one amino acid/protein synthesis one at a time. When the channel doesn't function, we know that the amino acid was functional.

However, this becomes a very expensive and inelegant method.

What are the other methods/techniques that we should look at to solve the problem?

Exact challenge now is to bring down the suspected list of amino acids from an initial, say, 10 to 4.

I guess this means there is some technique that uses assumptions and broad categorization rather than confirmatory tests like the site directed mutagenesis above.
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Postby JackBean » Tue Oct 05, 2010 10:26 am

well, you could get some clue, if e.g. some part of the protein was deleted and it worked/didn't work
The other way is some homology modeling...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Potassium Channels - Determining the functional amino acids!

Postby dharavsolanki » Tue Oct 05, 2010 4:56 pm

Hi!

Thanks. But I really do not comprehend this very well. Agreed that the clues would be obtained by seeing if after the mutants are obtained, whether they work or do not work.

But, how do we know if they are working or not? For example, I was searching for some information on the web about using site directed mutagenesis to pin point the functional amino acids.

[[[ Q1 : Is this called sequential mutagenesis? ]]]

I came across this - http://www.cell.com/retrieve/pii/0092867489909422 - its an article on site directed mutagenesis on HIV. It says something about "Quasispecies being different" BUT "in-vitro sample were indistinguishable."

This has got me confused.

Also, my report has to revolve around positive and negative controls.

[[[ Q2 : How will you go about reducing the ten suspects to six, and what techniques will be used in the laboratory? ]]]

Q3 : What protein structures of the selective potassium channel can be similar to some other known protein structure. Actually, I couldn't grasp the idea why homology modelling should be used here. I looked up on it and all I can get from it is that it can be used to predict or approximate structure of an unknown protein when a simlar gene sequence as the corresponding sequence of the protein is known, and that the gene latter protein structure is known to us.

If you could answer these three questions, maybe I could go a little bit forward.
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Postby dharavsolanki » Tue Oct 05, 2010 4:59 pm

"It says something about "Quasispecies being different" BUT "in-vitro sample were indistinguishable." This has got me confused."

What I mean to say here is that I am confused as to how two different species can be identified but still have the same in vitro extracts. Or does it mean that the difference is made in something which is not an extractable product? What is the significance of this statement and what are the possibilities that it suggests?
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Re: Potassium Channels - Determining the functional amino acids!

Postby dharavsolanki » Tue Oct 05, 2010 5:22 pm

Additional Detail : We have been told to do the problem on neuronal cells, as it eases up some things; neuronal cells being the largest mammalian cells!
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Postby JackBean » Wed Oct 06, 2010 10:12 am

I think the neoronal cells are chosen, because they contain lot of channels, not predominantly because of the size.

The positive control can always be your WT protein or silence mutation, negative control could be like empty vector, depending on how will you measure it.

http://en.wikipedia.org/wiki/Potassium_ ... #Structure
http://www.biolib.cz/en/main/

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