W. John Haynes

Yeast screens show aromatic residues at the end of the sixth helix anchor transient receptor potential channel gate

Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance, the arrangement and roles of the amino acids that constitute the TRP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca2+. After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca2+, indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one α-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed.

Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure–function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

Expression of the green fluorescent protein in Paramecium tetraurelia.

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P.tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P.caudatum telomerase RNA can participate in telomere synthesis when expressed in the P.tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P.tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P.tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P.caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

Toward Cloning Genes by Complementation in Paramecium

Conventional methods of gene cloning by complementing mutant defects is made difficult by the 800 ploidy of the Paramecium macronucleus. However, this nucleus is some 30 microns in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for massive injection of engineered DNA fragments and their maintenance in the transformed descendant. If a genomic DNA fraction injected into a mutant macronucleus effects complementation, it should be possible to sort a fractional library to isolate the complementing gene. Here, we investigated four aspects of establishing this method for general use. First, using the cloned CAM gene as a test case, we further investigated transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgene, even when it is of a recessive allele, cam2, which has a missense mutation but produces a partially functional protein. Second, we examined the copy number of the transgene established in cells of older clonal age and discussed the likely dilution of the transgene in younger descendants of the injected cell. Third, we showed that the degree of phenotypic reversion is correlated with the transgene product, the cam2 calmodulin protein in the cell. Fourth, we extended the investigation to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong phenotypic reversions in several pawn mutants, setting the stage for cloning these Ca(2+)-channel related genes. The general usefulness of this method in cloning genes that complement recessive alleles and current limitations of this method in dealing with dominant alleles are assessed and discussed.

Microbial K+ channels

A survey of the currently available genomes shows that K+ channels are found in most free-living bacteria, archaea, and protists, indicating early evolution before the divergences of the three major domains of life. Though vertically descended from the primordial K+ channels, more recent lateral

PAK Paradox: Paramecium Appears To Have More K+-Channel Genes than Humans

ABSTRACT K+-selective ion channels (K+ channels) have been found in bacteria, archaea, eucarya, and viruses. In Paramecium and other ciliates, K+ currents play an essential role in cilia-based motility. We have retrieved and sequenced seven closely related Paramecium K+-channel gene (PAK) sequences by using previously reported fragments. An additional eight unique K+-channel sequences were retrieved from an indexed library recently used in a pilot genome sequencing project. Alignments of these protein translations indicate that while these 15 genes have diverged at different times, they all maintain many characteristics associated with just one subclass of metazoan K+ channels (CNG/ERG type). Our results indicate that most of the genes are expressed, because all predicted frameshifts and several gaps in the homolog alignments contain Paramecium intron sequences deleted from reverse transcription-PCR products. Some of the variations in the 15 genomic nucleotide sequences involve an absence of introns, even between very closely related sequences, suggesting a potential occurrence of reverse transcription in the past. Extrapolation from the available genome sequence indicates that Paramecium harbors as many as several hundred of this one type of K+-channel gene. This quantity is far more numerous than those of K+-channel genes of all types known in any metazoan (e.g., ∼80 in humans, ∼30 in flies, and ∼15 in Arabidopsis). In an effort to understand this plurality, we discuss several possible reasons for their maintenance, including variations in expression levels in response to changes in the freshwater environment, like that seen with other major plasma membrane proteins in Paramecium.

An exchanger-like protein underlies the large Mg2+ current in Paramecium

There are very few molecules known to transport Mg2+ in eukaryotes. The membrane of Paramecium tetraurelia passes a large Mg2+-selective current and exhibits a corresponding backward swimming behavior. Both are missing in a group of mutants called eccentric. By sorting an indexed WT genomic library through microinjection into the macronucleus, we have isolated a DNA fragment that complements the eccentric mutations. The Mg2+ currents and behavior are restored fully in the transformed cells. Surprisingly, the conceptually translated protein is not homologous to any known ion channel but instead has some similarity to K+-dependent Na+/Ca2+ exchangers. Exchangers are either electrically silent or only pass very small and slow currents compared with ion-channel currents. In light of recent ion-channel crystal structures and considering the need to have narrow ion-selective filters, we speculate on how an exchanger might evolve to show channel-like activities in special circumstances. The significance of finding the molecular basis of a Mg2+-specific pathway is also discussed.

A Comparison of Internal Eliminated Sequences in the Genes that Encode Two K+-Channel Isoforms in Paramecium tetraurelia

We examined both the somatic (macro‐) and the germinal (micronuclear) DNAs that encode two K+‐channel isoforms. PAK1 and PAK11, in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5′‐most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206‐bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K+‐channel isoforms.

Microbial TRP Channels and Their Mechanosensitivity

Publisher Summary The pioneering phenotype‐to‐gene approach that dissects biological pathways has led to transient receptor potential (TRP) channels. Follow‐up examinations of these TRPs and their homologues constitute the bulk of current research on this superfamily of fascinating channels that are central to many aspects of animal physiology. Beyond animals, TRP genes can be found in Paramecium, Tetrahymena, Dictyostelium, Trypanosoma, Leishmania , and other protists, as well as most species of fungi. Thus, these channels and their basic mechanisms have evolved long before the appearance of multicellular animals. The similarities of the invisible molecules have evolved to distinguish only visible size, shape, and movement. Man, chimp, redwood, and Escherichia coli , though different in size shape, and movement, are made of the same clay. Very few macromolecules were invented since the appearance of multicellular animals, relative new comers in evolutionary history. The chapter summarizes the presence of TRP channels in protists and fungi. If all TRPs have the same origin and the conserved TM5–TM6 region holds the secret, then any TRP channel should be equally appealing as the subject of investigation.