Voltage-dependent sodium channels are believed to have evolved from calcium channels at the origin of the nervous system. A search of the genome of a single-celled choanoflagellate (the sister group of animals) identified a gene that is homologous to animal sodium channels and has a putative ion selectivity filter intermediate between calcium and sodium channels. Searches of a wide variety of animal genomes, including representatives of each basal lineage, revealed that similar homologs were retained in most lineages. One of these, the Placozoa, does not possess a nervous system. We cloned and sequenced the full choanoflagellate channel and parts of two placozoan channels from mRNA, showing that they are expressed. Phylogenetic analysis clusters the genes for these channels with other known sodium channels. From this phylogeny we infer ancestral states of the ion selectivity filter and show that this state has been retained in the choanoflagellate and placozoan channels. We also identify key gene duplications and losses and show convergent amino acid replacements at important points along the animal lineage.eumetazoan | inactivation gate | pore motif

Voltage-gated ion channels are large transmembrane proteins that enable the passage of ions through their pore across the cell membrane. These channels belong to one superfamily and carry pivotal roles such as the propagation of neuronal and muscular action potentials and the promotion of neurotransmitter secretion in synapses. In this review, we describe in detail the current state of knowledge regarding the evolution of these channels with a special emphasis on the metazoan lineage. We highlight the contribution of the genomic revolution to the understanding of ion channel evolution and for revealing that these channels appeared long before the appearance of the first animal. We also explain how the elucidation of channel selectivity properties and function in non-bilaterian animals such as cnidarians (sea anemones, corals, jellyfish and hydroids) can contribute to the study of channel evolution. Finally, we point to open questions and future directions in this field of research. KEY WORDS: Voltage-gated ion channels, Animal evolution, Ion selectivity Introduction: the superfamily of voltage-gated ion channelsThis review aims to cover and clarify the evolution and diversification of metazoan voltage-gated ion channels, particularly at the beginning of multicellularity in the lineage leading to Metazoa and at the emergence of nervous systems. Voltage-gated ion channels are imperative for neuronal signaling, muscle contraction and secretion, and are thought to play a critical role in the evolution of animals (Hille, 2001). Nonetheless, these channels are also found in prokaryotes and viruses, although their roles in these organisms are largely unknown (Martinac et al., 2008;Plugge et al., 2000). The superfamily of voltage-gated ion channels is characterized by the ability to rapidly respond to changes in membrane potential (hence 'voltage-gated'), which results in selective ion conductance. This ion channel superfamily includes voltage-gated potassium channels (K V s), voltage-gated calcium channels (Ca V s) and voltage-gated sodium channels (Na V s). Their α-subunits are composed of four domains (DI-IV), with each domain containing six transmembrane segments (S1-S6; Fig. 1) (Noda et al., 1984;Noda et al., 1986;Guy and Seetharamulu, 1986;Tanabe et al., 1987). Voltage-dependent activation is enabled by conserved positively charged residues at every third position in S4 (voltage sensor) of the four domains, which move outwards upon changes in membrane potential (Noda et al., 1984; Catterall, 1986;Guy and Seetharamulu, 1986), inducing a conformational change that results in opening of the channel pore (Armstrong and Bezanilla, 1974;Stühmer et al., 1989;Papazian et al., 1991;Yang et al., 1996). The pore is formed by the segments S5 and S6, and the selectivity to specific ions is enabled by the selectivity filter, which is composed of conserved residues, specific for the ion conducted by the channel, and these residues are situated at the pore-lining loops (p-loops) connecting S5 to S6 in the four domains ( ...

Calmodulin (CaM) is a Ca 2+ -sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca 2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca 2+ -dependent manner. We do not fully understand how CaM performs its role as a highfidelity signal transducer for more than 300 target proteins, but diversity among its four Ca 2+ -binding sites, called EF-hands, may contribute to CaM's functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca 2+ signals using four specialized EFhands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.calcium signaling | EF-hand | structure | evolution | protein

Multicellularity has evolved multiple times, but animals are the only multicellular lineage with nervous systems. This fact implies that the origin of nervous systems was an unlikely event, yet recent comparisons among extant taxa suggest that animal nervous systems may have evolved multiple times independently. Here, we use ancestral gene content reconstruction to track the timing of gene family expansions for the major families of ion-channel proteins that drive nervous system function. We find that animals with nervous systems have broadly similar complements of ionchannel types but that these complements likely evolved independently. We also find that ion-channel gene family evolution has included large loss events, two of which were immediately followed by rounds of duplication. Ctenophores, cnidarians, and bilaterians underwent independent bouts of gene expansion in channel families involved in synaptic transmission and action potential shaping. We suggest that expansions of these family types may represent a genomic signature of expanding nervous system complexity. Ancestral nodes in which nervous systems are currently hypothesized to have originated did not experience large expansions, making it difficult to distinguish among competing hypotheses of nervous system origins and suggesting that the origin of nerves was not attended by an immediate burst of complexity. Rather, the evolution of nervous system complexity appears to resemble a slow fuse in stem animals followed by many independent bouts of gene gain and loss.ancestral genome reconstruction | ASIC/ENaC | Cys-loop receptor | ionotropic glutamate receptor | potassium channel

Correctly estimating the age of a gene or gene family is important for a variety of fields, including molecular evolution, comparative genomics, and phylogenetics, and increasingly for systems biology and disease genetics. However, most studies use only a point estimate of a gene’s age, neglecting the substantial uncertainty involved in this estimation. Here, we characterize this uncertainty by investigating the effect of algorithm choice on gene-age inference and calculate consensus gene ages with attendant error distributions for a variety of model eukaryotes. We use 13 orthology inference algorithms to create gene-age datasets and then characterize the error around each age-call on a per-gene and per-algorithm basis. Systematic error was found to be a large factor in estimating gene age, suggesting that simple consensus algorithms are not enough to give a reliable point estimate. We also found that different sources of error can affect downstream analyses, such as gene ontology enrichment. Our consensus gene-age datasets, with associated error terms, are made fully available at so that researchers can propagate this uncertainty through their analyses (geneages.org).