Molecular Biology and Genetics of Calmodulin in Paramecium

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This proposal is an investigation of calmodulin in Paramecium. It will extend the previous observation that a point mutation in calmodulin causes the Paramecium to swim backward. Calmodulin is involved in calcium-regulated potassium channels and cell viability in this organism. Other mutant calmodulins will be prepared and their sensitivity to barium ions will be examined; the mutant cam1 has been shown to have increased sensitivity to barium, which can replace calcium in its four binding sites. Intergenic suppressors of the calmodulin mutants cam1 and cam 11 will be sought, to try to find other genes involved in the regulation of ion channels and cell viability. Intragenic and intergenic suppressors which affect activation of the calcium-dependent potassium channel differentially from the barium sensitivity will be sought. Proteins which bind wild-type Paramecium calmodulin better than its mutant calmodulins will be searched for in mammalian cells, and the genes coding for these proteins will be isolated. Calmodulin is a protein which is present in all cell types which have been examined. It binds calcium and is involved in regulation of many aspects of cellular function. Among these are the regulation of channels which permit entry of ions into cells and regulation of the cell cycle. In the protozoan Paramecium, a mutation in a single amino acid has been shown to cause the organism to swim backward. In addition, this mutation increases the sensitivity of the organism to barium, which can substitute for calcium at specific binding sites. The proposal involves a search for additional calmodulin mutants by a variety of techniques, a search for intergenic suppressors of the proteins, and a search for proteins in mammalian cells which bind differently to wild-type and mutant calmodulins from the protozoan. Proteins which do not affect the activation of the calcium-dependent potassium channel but do affect the ability of calmodulin to bind barium will also be sought. The study should increase understanding of how calmodulin regulates cell processes.