Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM)

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.

Paramecium tetraurelia has the shortest known introns as its standard intron length. Sequenced introns vary between 20 and 33 nucleotides in length. The intron sequences were discovered in genomic sequences coding for a variety of different proteins, including phosphatases, kinases, and low-molecular weight GTP-binding proteins. All intron sequences begin with the conserved dinucleotide GT and end with the conserved dinucleotide AG. The sequences are more AT rich than the Paramecium coding sequences. The identified sequences were confirmed as introns by sequencing several cDNA fragments. We report here analysis of the characteristics of 50 separate introns, including size, base composition, and a consensus sequence.

Two mutants of Paramecium tetraurelia, called "pantophobiacs," were found to lack most of the slow Ca2+-induced K+ outward current. Passive properties, the transient Ca2' inward current, and the fast depolarization-induced K+ outward current remain normal. The mutant defect reduces the ability to shut off a normal, excited state of the membrane and results in repeated, long backward swimming instead of the wild-type jerks in response to a variety of ions, to heat, and to touch.Certain mutations are known to affect the nervous system and the behavior of animals (1). In Drosophila (2) and Paramecium (3-7), induced mutations can affect the electric properties of the excitable membranes. Because Paramecium is a large cell and therefore suitable for voltage-clamp experiments, several mutations have been shown to alter the currents through different ion channels (8-11). These mutations have been used as blockers to dissect the pathway of electrogenesis and behavior (4-6, 9, 12-15) and to provide the null controls in the measurements of individual ion currents (14-16) or fluxes (17-19). These mutations also have been used as markers in the search for the gene products involved in the ion-channel functions (20)(21)(22).A paramecium is much like a neuron with several kinds of ion channels differing in their properties and locations. It has at least seven kinds of ion channels with different triggering mechanisms, on-kinetics, off-kinetics, and ion selectivities, some of which are located in the somal membrane and others in the ciliary membrane (23,24). The currents through these channels can be separated by standard physiological methods, by the use of mutants (9,(13)(14)(15), and by deciliation (25,26). The Ca2+-induced K+ current is among the seven kinds of membrane currents studied to date. In paramecia, a step depolarization with a voltage clamp induces a transient Ca2+ inward current, followed by a complex K+ outward current. The Ca2+-induced portion of this complex has been estimated by subtracting the outward current of a pawn mutant, which lacks the Ca2+ current and the Ca2+-induced currents, from the outward current of the wild type. The Ca2+-induced K+ current thus isolated activates slowly, reaching its plateau seconds after depolarization (15

We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (>55% between subfamilies, >94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

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