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Our
long term goals are to identify and characterize proteins that may
be regulating ion channel
activity in Paramecium. Since a number of potential regulators
have been cloned, our immediate task is simply to screen them using
antisense technology. One class of regulators that we have cloned,
the protein phosphatases, are important regulatory proteins that
help coordinate other protein activities in the cell. My data indicates
that a particular type of protein phosphatase, called protein
phosphatase 1 (PP1), regulates the activity of one or both potassium
channels present in Paramecium (the voltage dependent and the calcium
dependent potassium channels). When these channels are not opened
properly, the cell swims backwards for long periods of time (in
the appropriate test solution). In my proposed research leave, I
want to further characterize the role that PP1 plays in K+ channel
regulation. Interestingly, there are two nearly identical genes
that have both been affected by my antisense treatment. Thus one
of my first goals is to determine if these genes are functionally
redundant or have different roles to play in the cell.
The
complete collection of PP1 isoforms that have been identified and
cloned in Paramecium will be 'sorted' as to their role in the regulation
of Paramecium electrophysiological processes using loss-of-function
and gain-of-function approaches. It is essential to unequivocally
determine which isoforms are involved so that subsequent experiments
are precise and focused upon the appropriate targets.
1.
Loss of function approach. Antisense techniques and one
of two 'gene knockout' approaches will be utilized to provide
an initial sorting of PP1 isoforms as to their role in the regulation
of Paramecium swimming behavior.
2.
Gain of function approach. This approach will verify and
extend characterizations made in Part 1 above and if necessary,
as a means to confirm negative results obtained in those experiments.
It relies on the observation that okadaic acid (OA) is a PP1-specific
inhibitor in Paramecium and does not inhibit Paramecium phosphoprotein
phosphatase type 2A (PP2A). A molecular genetic approach will
be used to generate okadaic acid resistant mutants to identify
which isoforms need to be altered to confer resistance with regards
to its effects upon swimming behavior and/or cell death.
Last
updated
July 18, 2001
Webmaster Dean Fraga.
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