Karl Peter Giese
p.giese@ucl.ac.uk
Department of Anatomy &
Developmental Biology, University College London, UK.
Gene targeting allows the
introduction of desired point mutations into genes. Three alternative strategies
(Hit and Run procedure, tag and exchange strategy, Pointlox procedure)
can be applied to generate targeted point mutants. These strategies will
be illustrated and their pros and cons will be discussed.
Targeted point mutants are
powerful tools for studying the role of protein modulation in learning
and memory ( L & M) and they can adequately model certain inherited
L & M disorders. Additionally, phenotypic comparisons between targeted
point mutants and corresponding null mutants can deepen insights into L
& M mechanisms. The phenotype of the a CaMKIIT286A mutants
will be discussed to illustrate most of these issues.
Autophosphorlylation at
threonine-286 (T286) of the a -isoform of the Ca2+/calmodulin-dependent
protein kinase II (a CaMKII) leads to an activity switch. This activity
switch had been suggested to be central for synaptic plasticity and memory.
Since the T286A mutation (threonine-286 changed to alanine) blocks the
activity switch by leaving the kinase in its basal activity state, the
generation/analysis of the a CaMKIIT286A mutants was the only
approach to test the "switch hypothesis". Consistent with the switch hypothesis,
the a CaMKIIT286A mutants are deficient for NMDA receptor-dependent
LTP in the hippocampal CA1 region, have unstable place cells and are impaired
in spatial learning in the water maze. The LTP deficits of the a CaMKIIT286A
mutants contradict the phenotype interpretation of transgenic mice over-expressing
constitutively active a CaMKII (a CaMKIIT286D). This controversy
will be discussed.
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