Dr. Ching Kung requests five years of support to continue his studies on the role of calmodulin in the swimming behavior of the ciliate, Parmecium. The swimming behavior of Paramecium is regulated by the activities of membrane ion channels. These ion channels are in turn regulated by calcium and by the calcium binding protein calmodulin. Each calcium action potential induces a reversal of the ciliary motion and a burst of backwards swimming. The Na+ current sustains the action potential and increases the backwards swimming. Outward K+ currents tend to end the action potential. In 1983, Dr. Kung and his collaborators isolated a Parmecium mutant that they termed """"""""pantophobiac"""""""" (pnt); it swims backwards for longer periods than do wildtype cells. This behavioral phenotype was the result of a mutation in the single calmodulin gene. Mutants that have extended or short backward swimming bouts were also isolated. Rather surprisingly, mutants that swim backwards for shorter periods (fast-2) also were in the calmodulin gene. While the pnt mutants are in the carboxy terminal part of the protein, the fast-2 mutants are in the amino terminal lobe. When the currents are investigated, the C-terminal mutants are missing or have reduced K+ current and the N-terminal mutants have reduced Na+ current. This segregation of mutant phenotypes and location is based on 13 mutations and none of the 13 tested fell into the central region of the protein. These observations have lead to the hypothesis that will tested further in this proposal that the calmodulin molecule is bifunctional. One end can interact with one set of molecules and the other end can interact with different molecules. Dr. Kung and his collaborators have tested whether Paramecium calmodulin can activate calcineurin, which is a calmodulin dependent protein phosphatase. They find that wildtype and C-terminal mutants can activate it, but N- terminal mutants cannot. This proposal has five specific aims. The partitioning of mutant alleles to the two ends will be examined further by the isolation of additional pantophobic and fast-2 mutants. Mutations generated in vitro will also be examined. The Paramecium calmodulin protein has been crystallized at 1.8 A resolution and they will examine the location of the mutants on the crystal structure with the idea that specific surfaces may be affected in the two types of mutants. In later years, mutant calmodulins will be examined by X-ray crystallography and NMR. In the second aim, the bifunctionality of calmodulin will be tested in collaboration with other laboratories that have assays for calmodulin- activated enzymes. These include calcineurin, red cell C++-ATPase, adenylate cyclase, myosin light chain kinase, Ca-calmodulin dependent protein kinase, and phosphodiesterase. In the third aim, they will attempt to clone two calmodulin-binding membrane proteins that they have identified in the last grant period. In a similar vein, they will finish isolating and characterizing homologs of K+ and Na+/Ca++channel genes. In a fourth aim, attempts will be made to improve transformation so that behavioral mutants can be rescued by transformation and complementation. In the final goal, a procedure known as PAJAMAS will be used to develop a method for depleting the macronucleus of a particular sequence so that reverse genetics can be done in Paramecium.
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