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PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.

Four mutant strains of Paramecium tetraurelia with a reduced ability to respond behaviorally to Mg2+ have been isolated. Voltage-clamp analyses showed that their Mg2+ insensitivity is associated with a reduced Ca(2+)-dependent Mg2+ current. The four mutants, which have been doubled "eccentric," result from recessive mutations in two unlinked loci, xntA and xntB. Further analysis of xntA1 showed it to be unlinked to any of the behavioral mutants of P. tetraurelia described previously, but it is allelic to d4-521, a "K(+)-resistant" strain, and d4-596, a "Ba(2+)-shy" mutant. The varied pleiotropic effects of xntA1, which include increased resistance to Ni2+ and Zn2+ poisoning, suggest that the locus encodes a central regulator of cell function in Paramecium.

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.

Paramecium aurelia is chosen as the material for a genetic dissection of the excitable membrane. The simple innate behavior makes the behavioral analyses easy and mutant screening possible. Autogamy, which leads to complete homozygosity, guarantees the expression of recessive mutations. The size of these giant cells allows intracellular recording in electrophysiological studies.--Some two hundred lines of behavioral mutants are isolated. Several of them are studied in detail genetically, behaviorally as well as electrophysiologically. Mutants devoid of proper active electrogenesis are now available. Some of them are shown to be temperature-dependent. They have a slightly aberrant electric pattern when grown at the permissive temperatures and lose their excitability completely when cultured at the restrictive temperatures.--Systematic modifications of the membrane excitation process is demonstrated in this interdisciplinary study. Three unlinked mutations are shown to block the generation of Na-triggered depolarizations at different positions resulting in three different altered forms of electrical activities. The bioelectric profiles of 25 types of single or double mutants are now known. The search for the relevant gene products on the surface membrane is now underway.

Six mutants of Paramecium tetraurelia with curious "Paranoiac" phenotypes have been isolated and examined. Instead of the normal transient avoiding reactions in Na+ solution, these mutants show "violent avoidances"--backing continuously for 10 to over 60 sec. This behavior corresponds to prolonged membrane excitation excitation.--Genetic analyses establish five genic loci at which mutations give the "Paranoiac" phenotype. Close linkage between two of these genes occurs. Allelic variants are found for two of the genes. In one case, the two alleles determine very different behavioral phenotypes ("Paranoiac" and "fast-2"). These results show that the mechanism(s) which shuts off excitation in the wild-type membrane is (are) complex, but in the future may be fruitfully pursued in mutants which are defective.

Dancers are a group of mutants in Paramecium tetraurelia whose Ca2+ current inactivates poorly and are likely to be defective in the structure of their Ca2+ channels. These mutants show prolonged backward swimming in response to K+ and Ba2+ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F1 generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca2+ channel.

Heat-sensitive Pawn (ts Pawns) of Paramecium aurelia behaved normally when grown at 23 degrees but failed to avoid strong stimuli at 35 degrees . Four of the five ts Pawn lines tested were found to be allelic at a locus known also to carry temperature-independent Pawn mutations. The fifth ts Pawn line complemented all the conditional and unconditional Pawn mutants of the two known loci. This result, together with the patterns of F(2) segregation from various crosses, suggested the existence of a third Pawn locus. An additive effect of the unlinked ts Pawn genes was observed. These findings and the significance of ts Pawns as experimental material in behavioral and physiological research are discussed.