NEW NOTE-LETHAL CALMODULIN MUTATIONS IN PARAMECIUM - A STRUCTURAL ANDFUNCTIONAL BIPARTITION HYPOTHESIS

The mechanisms by which calmodulin coordinates its numerous molecular targets in living cells remain largely unknown. To further understand how this pivotal Ca2+-binding protein functions in vivo, we isolated a nd studied nine new Paramecium behavioral mutants defective in calmodu lin. Nucleotide sequences of mutant calmodulin genes indicated single amino-acid substitutions in mutants cam(4)(E104K), cam(5)-1 (D95G), ca m(6) (A102V), cam(7) (H135R), cam(14)-1 (G59S) and cam(15) (D50G). In addition, we encountered a second occurrence of three identified subst itutions; they are cam(1)-2 (S1O1F), cam(5)-2 (D95G) and cam(14)-2 (G5 9S). Most of these mutational changes occurred in sites that have been highly conserved throughout evolution. Furthermore, most of these cha nges were not among the amino acids known to interact with the basic a mphiphilic peptides of calmodulin targets. Consistent with our previou s finding [Kink, J. A., Maley, M. E., Preston R. R., Ling, K.-Y., Wall en-Friedman, M. A., Saimi, Y. and Kung, C. (1990) Cell 62, 165-174], m utants that under-reacted to certain stimuli (allele number above 10) had substitutions in the N-tenninal lobe of calmodulin, and those that over-reacted (below 10) had substitutions in the C-terrninal lobe. No mutations were found in the central helix that connects the lobes. Th us, through undirected in vivo mutation analyses of Paramecium, we dis covered that each of the two lobes of calmodulin has a distinct role i n regulating the function of a specific ion channel and eventually the behavior of Paramecium. We, therefore, propose a hypothesis of functi onal bipartition of calmodulin that reflects its structural bipartitio n.