Letter ; ), ), leak + Gibberella ons, please )andhavesome Hong et al. 2010 Swayne et al. 2009 ; ; fungal four-domain channels ), and circadian rhythms in flies itive channels in the eukaryotic NALCN channels diverged from Paidhungat and Garrett 1997 ). These channels will be called channels begin to open. NALCN Lee et al. 1999 Lu et al. 2009 Liu et al. 2006 v ( ). As they leak sodium into the cell, Ren 2011). These fungal channels are ) channels, which underlie impulse con- v ; ). Fungal calcium channels have been impli- channels ( ). v ). ine Biological Laboratory, Woods Hole, Lu et al. 2007 1,2,3 and Na Hallen and Trail 2008 v ( NALCN channels maintain and regulate firing rates in It has been shown previously that NALCN channels di- Hong et al. 2010 Pierce-Shimomura et al. 2008 Nash et al. 2002 Hong et al. 2010 fungal calcium channels here for simplicity, but there are and ascospore dischargezeae in the plant pathogen cated in matingcalcium-store in restoration yeast inCryptococcus ( the neoformans meningitis-causing fungus rhythmically firing neurons bypotential modulating ( neuronal resting the membrane becomes depolarized frompotential the set very by negative thethe efflux threshold of at which potassium Na and moves toward strongly selective forinsensitive calcium but and like( rely NALCN on are voltage an accessory protein for gating Lu et al. 2010 verged from voltage-gated channels beforeof the diversification Ca channels may therefore be thoughttheneuron:themoreNALCNchannelsareopen,themore of as affecting the gainlikely of an input is towill initiate continue firing to fire). (or Although that theytheir a are insensitive rhythmic open to neuron state voltage, canneurotransmitters be and by affected calcium, by and theyproteins the rely for presence on their accessory of function ( various ( ( ), and sodium (Na similarities to the lone family( of v vance Access publication July 19, 2012 3613 cellular relatives. We show that ve “sodium leak” channel, have garnered a growing interest. This ate behavior across the tree of life. These proteins regulate the voltage-gated and voltage-insens v ty for Molecular Biology and Evolution. All rights reserved. For permissi ily are opened by changes in voltage, however. NALCN (NA v /Ca Cai v Hille ). Ca onal Biology and Bioinformatics, University of Texas online; and Harold H. Zakon family just 1 v leak channel Hille 2001 + Liebeskind et al. ). ; channels, but are nce of fungi and animals and that the closest relatives of NALCN channels are fungal v ve Molecular Biology and Evolution, Mar Caenorhabditis elegans ) channels that mediate v Cai 2012 e-gated potassium, calcium (Ca and Na Ren 2011) such as breathing v David M. Hillis, ,1 Supplementary Material E-mail: [email protected]. , ), crawling in Emes and Grant 2011 channels arose from the Ca ; Torruella et al. 2011). The most obviously v ), and sodium (Na ; v Andrew Roger 29(12):3613–3616 doi:10.1093/molbev/mss182 Ad NALCN, Cch1, maximum likelihood, pore motif. channels have four domains, each with six trans- Lu et al. 2007 channels arose from single domain potassium chan-

v v Cai 2012). We continue this project, focusing here on

The voltage-gated ion channel family includes the potas- Sodium leak channels, or NALCN (NA The Author 2012. Published by Oxford University Press on behalf of the Socie Section of Neurobiology, UniversityJosephine of Bay Texas Paul Center for Comparati Section of Integrative Biology and Center for Computati supplementary fig. S1 2011; the evolution of opisthokont four-domain ion channels. sium, calcium (Ca Massachusetts *Corresponding author: Associate editor: 2001)andthatNa nels by internal duplication at the base of eukaryotes ( the neural code by creating action potentials ( channel nonselective) channels, which encode a voltage-insensiti study examines the phylogenetic relationship among Na e-mail: [email protected] ß Mol. Biol. Evol. and Na Abstract Proteins in the superfamily of voltage-gated ion channels medi 3 Calcium Channels in an Ancient,Benjamin Voltage-Insensitive J. Liebeskind,* 1 Clade 2 movement of ions across cell membranesof by opening this and superfamily closing a are central the pore that voltag controls ion flow. The best-known members Phylogeny Unites Animal Sodium Leak Channels with Fungal membrane segments. Each domain hasthe a fifth pore loop and between sixthamino acids segments, that determines forming ion a selectivity.that It Ca pore is hypothesized motif of four nonselective), are four-domainthesamesixtrans-membranesegmentdomainastheir channels that arebetter studied built relatives, on the Ca before the origin of opisthokonts ( duction in nerve and muscle. Not all members of this fam distinguishing feature of animals isbehavior the in elaboration adults, of facilitated motile bymuscle. Recent the studies evolution have of used nervesphylogenetics comparative to and genomics examine the and history of nervousand system show genes how this history bears on eukaryotic diversity ( Parfrey et al. 2011 and Clapham 2011 The eukaryotic supergroup Opisthokonta containskingdoms two large with very( different life styles: fungi and animals group Opisthokonta, which includes animals, fungi,voltage-gated and channels their before uni the diverge calcium channels, which they functionally resemble. Key words: voltage insensitive. NALCN channels havenumerous been rhythmic implicated in behaviors ( in mice ( Liebeskind et al. . doi:10.1093/molbev/mss182 MBE other calcium channels in fungi that are not homologous to in the third domain of NALCN channels is thought to render animal four-domain channels (Zelter et al. 2004). the channels nonselective among cations (Lu et al. 2007) In this study, we sought to clarify the phylogenetic rela- becausesinglelysinesubstitutions in wild-type (E/E/E/E) Cav tionships between the major lineages of opisthokont channel pores eliminate selectivity for calcium over monova- four-domain ion channels. We use this phylogenetic informa- lent cations (Yang et al. 1993). It is therefore likely that tion to infer the historical timing of key amino acid replace- nonbilaterian NALCN channels actually function as cal- ments that may have had large-scale effects on opisthokont cium-permeable channels. These findings reinforce the view evolution. This study builds upon and synthesizes previous that changes in ion channel selectivity were major steps in the work, which identified the unique place of voltage-insensitive evolution of complex nervous systems in animals (Hille 2001; ion channels but did not place this information in the context Liebeskind 2011; Liebeskind et al. 2011). of opisthokont evolution (Paidhungat and Garrett 1997; Lee The earliest branching fungal calcium channel (Allomyces) et al. 1999; Ren 2011). also had an acidic pore motif (fig. 2), which suggests that the We used basic local alignment search tool searches to common ancestor of all voltage-insensitive channels had an identify NALCN homologs in some of the oldest animal lin- acidic pore and was permeable to calcium. The most diverse eages, including cnidarians (Nematostella vectensis), placozo- lineages of fungi (ascomycetes and zygomycetes) then fixed ans (Trichoplax adhaerens), and sponges (Amphimedon for polar uncharged amino acids (N or Q) in the first domain queenslandica). We also found four-domain fungal channels pore loop (fig. 2). Basidiomycetes are another diverse fungal in diverse fungal lineages, including the early-branching clade that was not sampled here but have an identical pore to Zygomycota (Phycomyces blakesleeanus,andMucor cir- their sister group, the ascomycetes (data not shown). Unlike cinelloides) and Blastocladiomycota (Allomyces macrogynus). animal Cav channels, fungal calcium channels with an N/E/E/E Fungal calcium channels and NALCN homologs were notably porearenotpermeabletosodiumevenintheabsenceof absent in single-celled opisthokont genomes. We aligned the calcium (Hong et al. 2010). Because fungal calcium channels new sequences with previously identified Cav and Nav chan- are necessary for survival in low-calcium environments in nels from animals, choanoflagellates, and the apusozoan pro- several fungal lineages, this may be adaptive (Liu et al. 2006; tist Thecamonas trahens (Liebeskind et al. 2011; Cai 2012), Hong et al. 2010). The fixation for N or Q in the pore accom- thought to be the sister group to opisthokonts (Torruella panied a loss of swimming zoospores in fungi at the blasto- et al. 2011). Support for a monophyletic apusozoan clade is cladiomycete/zygomycete boundary. This is notable because weak (Cavalier-Smith and Chao 2003), but we will refer to the calcium channels underlie mating in yeast and ascospore apusomonad Thecamonas as an apusozoan to be consistent bursting in Gibberella zeae (Fischer et al. 1997; Hallen and with the online database from which the sequence came Trail 2008) and may therefore be involved in mating behavior (supplementary materials, Supplementary Material online) in many other fungi. and with recent literature (Torruella et al. 2011). Thus, the early branching lineages of both NALCN and Phylogenetic analysis using maximum likelihood and fungal channels retained acidic motifs, but the most diverse Bayesian methods places fungal calcium channels and groups of sampled animals and fungi (bilaterians in animals, NALCN-like sequences within a well-defined clade to the and ascomycetes and zygomycetes in fungi) evolved different exclusion of voltage-gated Cav and Nav sequences (fig. 1, pore motifs early in their diversification. How these changes supplementary methods, Supplementary Material online). may have affected the evolution of animals and fungi will The topology was robust to model choice and estimation require characterization of channels that group close to the method and is consistent with known species trees (Torruella roots of these groups. et al. 2011). The voltage-insensitive clade split from the Characterized NALCN and fungal calcium channels are voltage-gated group that includes animal Cav and Nav chan- voltage insensitive (Lu et al. 2007; Hong et al. 2010), and all nels before the divergence of the fungal and animal lineages. channels in this clade had reduced numbers of Unlike Nav and Cav channels, which underwent several voltage-sensing residues relative to voltage-gated channels rounds of duplication in animals (Liebeskind et al. 2011; (supplementary fig. S2, Supplementary Material online). Zakon et al. 2011), NALCN channels were found in single This suggests that the homolog in the common ancestor copy in most species examined (fig. 1). The sponge A. queen- of animals and fungi was not voltage gated and that voltage slandica, the cnidarian N. vectensis, and the nematode C. insensitivity is therefore a shared, derived character of this elegans (Pierce-Shimomura et al. 2008) are exceptions to clade. Because both fungal calcium channels and NALCN rely this rule and each have two genes. This is notable because on accessory proteins for their function, it is also likely that neither sponges nor nematodes have Nav channels. The pres- this characteristic evolved at the base of the clade. Although ence of NALCN in all examined species has been noted pre- we found no obvious sequence similarity between the known viously in bilaterians (Ren 2011), and this finding extends this accessory proteins of NALCN and fungal calcium channels, it trend to nonbilaterians. seems likely that modulation by other proteins facilitated the Nonbilaterian NALCN channels do not have the same pore loss of voltage sensitivity in an ancestral channel and that the sequence as previously identified NALCN channels (E/E/K/E modulating proteins themselves have changed over time. or E/K/E/E). NALCN channels in Amphimedon, Trichoplax, Figure 1 is rooted at the midpoint. To get a more reliable and Nematostella have E/E/E/E in the pore, identical to rooting, we used voltage-insensitive and voltage-gated chan- high-voltage-activated Cav channels (fig. 2). The lysine (“K”) nels as queries to search nonopisthokont genomes for a

3614 Phylogeny of Opisthokont Four-Domain Ion Channels . doi:10.1093/molbev/mss182 MBE

FIG.1.Phylogenetic tree of opisthokont four-domain ion channels. NALCN and four-domain fungal calcium channels group together in a well-supported clade. Bootstrap proportions and posterior probabilities are reported for each branch. The cartoon on the right shows major groups of channels, including low- and high-voltage-activated Cav channels (LVA and HVA, respectively), Nav channels, NALCN, and four-domain fungal calcium channel. Hash marks on the left-hand tree denote branches that have been shortened for ease of display.

Lysine (K) Replacement Homo E/E/K/E Caenorhabditis E/E/K/E Bilateria Drosophila E/E/K/E } NALCN Nematostella E/E/E/E Trichoplax E/E/E/E Amphimedon E/E/E/E Saccharomyces N/E/E/E } Ascomycota Aspergillus N/E/E/E 2+ Fungal Ca Phycomyces Q/E/E/E Zygomycota Mucor Q/E/E/E} Allomyces E/D/E/D - Blastocladiomycota Thecamonas 3 E/D/E/E Polar Uncharged (N or Q) Replacement

FIG.2. Pore states mapped onto the voltage-insensitive subtree. We show two fixations for a polar, uncharged amino acid along the branches leading to ascomycetes and zygomycetes, but it is equally possible that either an asparagine (N) or a glutamine (Q) could have fixed in the common ancestor of these lineages (black circle) and changed to the other amino acid along one of the branches. Early branching lineages have pores with acidic residues (D or E). channel that diverged before the diversification of the chan- Supplementary Material online). However, rooting between nels represented in figure 1. Most major eukaryote lineages voltage-gated and voltage-insensitive channels, as in figure 1, have four-domain channels, many of which are hypothesized produces a more parsimonious pattern of gene loss in fungi to be calcium channels on the basis of their pore motifs than rooting with either voltage-gated group (one loss instead (Verret et al. 2010; Prole and Taylor 2011). We added 11 of two). Some nonopisthokont channels were identical to nonopisthokont sequences that had good coverage of taxa animal Cav channels in their pore sequence but had Nav- and channel types to the phylogeny in figure 1. like inactivation loop motifs (supplementary fig. S4, These sequences could not be reliably placed within the Supplementary Material online) (Smith and Goldin 1997). phylogeny, however, making root placement and the status of These enigmatic similarities cannot be adequately explained the Thecamonas channels uncertain (supplementary fig. S3b, at present but suggest a complicated evolutionary history.

3615 Liebeskind et al. . doi:10.1093/molbev/mss182 MBE

Our phylogeny suggests that an ancient loss of voltage Liebeskind BJ, Hillis DM, Zakon HH. 2011. Evolution of sodium channels sensitivity in a lineage of four-domain ion channels and key predates the origin of nervous systems in animals. Proc Natl Acad Sci amino acid replacements affecting ion selectivity in this line- USA.108:9154–9159. agewerebothfactorsinthediversificationoffungiandan- Liu M, Du P, Heinrich G, Cox GM, Gelli A. 2006. Cch1 mediates calcium imals. This phylogenetic information clarifies the evolution of entry in cryptococcus neoformans and is essential in low-calcium voltage-insensitive four-domain channels and suggests fungal Environments. Eukaryot Cell. 5:1788–1796. calcium channels as possible models for future NALCN Lu B, Su Y, Das S, Liu J, Xia J, Ren D. 2007. The neuronal channel NALCN research. contributes resting sodium permeability and is required for normal respiratory rhythm. Cell 129:371–383. Supplementary Material Lu B, Su Y, Das S, Wang H, Wang Y, Liu J, Ren D. 2009. Peptide neuro- Supplementary methods and figures S1–S4 areavailableat transmitters activate a cation channel complex of NALCN and Molecular Biology and Evolution online (http://www.mbe UNC-80. Nature 457:741–744. .oxfordjournals.org/). Lu B, Zhang Q, Wang H, Wang Y, Nakayama M, Ren D. 2010. Extracellular calcium controls background current and neuronal Acknowledgments excitability via an UNC79-UNC80-NALCN cation channel complex. Neuron 68:488–499. The authors thank Emily McTavish, Thomas Keller, April Wright, and Ammon Thompson for helpful comments on Nash HA, Scott RL, Lear BC, Allada R. 2002. An unusual cation channel the initial manuscript, and associate editor Dr. Andrew Roger mediates photic control of locomotion in Drosophila. Curr Biol. 12: 2152–2158. and two anonymous editors for thoughtful reviews. They also acknowledge the Broad Institute and their Origins of Paidhungat M, Garrett S. 1997. 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