A Hypothetical question!!!

Martin Leach leach at mbcrr.harvard.edu
Wed Apr 6 22:52:27 EST 1994

In article <2nv1jcINNld5 at dns1.NMSU.Edu>, smori at nmsu.edu (Shahram Mori)

> Dear netters,
> Pretend that you are studying K-channels. There are two types that can
> only be distinguished on their response to an inhibitor. The K channel
> that you are interested in is missing in one of the cell lines. Nothing is
> known about the protein and DNA sequence of either of these channels. How
> can you isolate and study this protein? The only one that I can think of
> is using the inhibitor in a column type setting and fish out the channel
> that I am interested in ( The protein that I want to study is the one that
> binds to the inhibitor). I would very much appreciate it if you had any
> suggestions as the best way to isolate and study this protein.
> Cheers.
> Shahram Mori
> Program in Molecular Biology
> Dept. of Chemistry
> Box 3C NMSU
> Las Cruces NM
> 88003
If this is your term paper then don't read the stuff below!!!

1. First characterise this channel.
   a. Ki for the inhibitor - activation voltages (if voltage gated)
   b. are g-proteins required to activate the channel etc...
   c. What is current going through this channel 
   d. Kinetics of activation/inactivation.

2. Once you know how to identify the channel then you can try isolating it.

3. Need a radiolabelled compound to follow the protein. Best to radiolabel
the inhibitor.

4. Isolate cell membranes from cells containing this receptor. And perform
receptor binding assay using radioligand (inhibitor)

5. To obtain an enriched protein prep. fractionate after eg. sucrose
density centrifugation after binding of radioligand.


7. If u wanted to clone the channel - 
   a. screen a cDNA library made from cells containing the channel - using
a probe made from another K channel. (using low stringency)
   b. Perform serial transfections of cDNA/expression constructs made from
the cell containing the channel - identify transfectants and look to see
which one has properties of K+ channel - isolate/identify construct in
successful transfectants - clone cDNA using DNA info from successful


phwew. out of breath now........i recommend number 6.

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
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