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First published online February 27, 2009
Journal of Experimental Biology 212, 761-767 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026971
Expression of a poriferan potassium channel: insights into the evolution of ion channels in metazoans
1 University of Alberta, Department of Biological Sciences, Edmonton, AB, Canada
T6G 2E9
2 University of California, Neuroscience Research Institute, Santa Barbara, CA
93117, USA
3 School of Integrative Biology, University of Queensland, Brisbane 4072,
Australia
4 University of Richmond, Department of Biology, Richmond, VA 23173, USA
Author for correspondence (e-mail:
lboland{at}richmond.edu)
Accepted 15 December 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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Key words: Kir, evolution, inwardly rectifying, ion channel, metazoa, sponge
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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;
Jiang et al., 2002;
Kuo et al., 2003;
Nishida et al., 2007) and in
mice (Nishida and MacKinnon,
2002). Knowledge of how membrane conduction properties changed
during the evolution of multicellularity would be aided by the comparative
analysis of K+ channels from phyla occupying basal positions.
Relationships among the earliest diverging animal groups are still not
clearly understood, with recent molecular evidence supporting one of
placozoans, ctenophores or sponges as the sister group to all other metazoans
(Dellaporta et al., 2006;
Dunn et al., 2008;
Srivastava et al., 2008). Most
phylogenies, however, place sponges (suspension-feeding animals lacking nerves
and muscle) as the most ancient multicellular metazoans. Analyses based on 18S
rDNA suggest that sponges arose first but are paraphyletic. This suggests that
a sponge-like animal gave rise to other metazoans
(Borchiellini et al., 2001;
Collins, 1998;
Sperling et al., 2007), and
that critical metazoan innovations might, therefore, be reflected in modern
sponges. Evidence for elements of the postsynaptic scaffold in sponge larvae
suggests this might be true (Sakarya et
al., 2007). Although sponges lack true nerves, they show many
types of non-nervous behavior. Aneural coordination in sponges involves
regulation of their feeding current by immediate flagella arrest
(Hexactinellida) (Leys and Mackie,
1997) or contraction of canals (Demospongiae, Calcarea)
(Elliott and Leys, 2007;
Nickel, 2004). In the former,
the mechanism is a calcium action potential
(Leys et al., 1999;
Leys and Eerkes-Medrano,
2006); in the latter it is thought to be by calcium waves
triggered by local release of signaling molecules
(Ellwanger and Nickel,
2006).
Intracellular recordings have not yet been obtained from any sponge due to
the difficulty of penetrating the complex glycocalyx surface coat and the thin
nature of cells and tissues lining the surface
(Litchfield and Morales, 1976;
Muller et al., 1998).
Patch-clamp recordings of cation channels have been made from the demosponge
Axinella polypoides (Carpaneto et
al., 2003; Zocchi et al.,
2001) but trivalent ions were required to obtain a seal and no
electrical conduction was found. Similar difficulty has been encountered in
patching glass sponge tissue and, given the extreme difficulty of collecting
and maintaining these deep-water animals, an ex situ approach is
considered to offer a better insight into ion channel function in sponges and
the evolution of electrical signaling in this phylum.
In this study, we report the cloning and functional characterization of the
first ion channels isolated from a sponge (Amphimedon
queenslandica, Demospongiae, Porifera). The newly available genome
project of the tropical demosponge A. queenslandica (formerly
Reniera sp.)
(http://www.jgi.doe.gov/sequencing/why/3161.html)
has facilitated progress in understanding the collection of metazoan
homologues present in the earliest animals. We have prioritized the study of
inward-rectifier potassium (Kir) channels for their basic structure and their
important role both in stabilizing the resting potential and regulating
cellular excitability. We present functional data on two clones, AmqKirA and
AmqKirB, whose properties were studied in the Xenopus oocyte
expression system. Sponge Kir channels share the phenotype and critical
residues that regulate strong inward rectification with vertebrate Kir2
channels, which are typically expressed in excitable cells including neurons
and skeletal and cardiac muscle cells
(Doupnik et al., 1995).
Phylogenetic comparison with other metazoan Kir channels suggests that Kir
channels in sponges, cnidaria and triploblastic metazoans each arose from a
single channel and that duplications arose independently in the different
groups. Diversification into the channel subfamilies from Kir1 to Kir7
occurred independently in the chordates.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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). For
RCA, cDNA was synthesized from 5 µg total RNA using Superscript III Reverse
Transcriptase Module (Invitrogen) with a 5'-phosphorylated OligodT
primer (Ellwanger and Nickel,
2006). Following purification with the QIAquick PCR purification
kit (Qiagen), full-length transcripts circularized with 150 units CircLigase
enzyme (Epicentre, Madison, WI, USA) in 1x Circligase Buffer with 50
µmol l–1 ATP. The reaction was incubated for 1 h at
60°C, inactivated at 80°C for 10 min and purified as above.
Circularized templates were amplified using 10 units of phi29 DNA polymerase
in 1x phi29 DNA Polymerase Reaction Buffer, supplemented with 200
µgml–1 bovine serum albumin, 200 µmol
l–1 dNTPs and 10 µmol l–1 random
heptamers, which were modified by 3'-phosphothiote linkages to make the
primers resistant to phi29 exonuclease activity. Amplification proceeded for
20 h at 30°C and was inactivated by incubation at 70°C for 10 min.
Putative ion channel sequences were assembled from unannotated trace files
of the A. queenslandica (formerly Reniera sp.) genome as
previously described (Sakarya et al.,
2007). Initial fragments were amplified using specific primers
designed against these sequences. The complete cDNA sequences were determined
using a combination of inverse PCR on RCA cDNA template
(Polidoros et al., 2006) and
RACE-PCR on GeneRACE-ready cDNA (Invitrogen GeneRacer kit). Products were
cloned into the PCR-4-TOPO (Invitrogen) vector and sequenced. Open reading
frames were amplified using primers modified with restriction sites to allow
ligation into pXT7 expression plasmid
(Dominguez et al., 1995). The
full sequence was confirmed from uncloned PCR products from at least two
independent PCR reactions, and clones free of PCR errors were selected for
expression.
Amino acid sequence and phylogenetic analysis
Sponge inward-rectifier sequences were aligned and compared with amino acid
sequences of both invertebrate and vertebrate inward-rectifier sequences
compiled from NCBI, Joint Genome Institute
(http://www.jgi.doe.gov/sequencing/why/3161.html)
and Ensembl databases. To determine pairwise identity values, AmqKirA and
AmqKirB amino acid sequences were each in turn aligned using LALIGN online
software
(http://www.ch.embnet.org/software/LALIGN_form.html)
and default parameters for global alignments. Multiple alignments were
generated using MUSCLE (Edgar,
2004). Alignments used for phylogenetic analysis were manually
adjusted in MacClade (Maddison and
Maddison, 2003) and a PERL script was used to remove characters
with more than 2% gaps to produce a trimmed alignment of 122 sequences and 280
positions. All data sets are available on request. The optimal tree topology
and Bayesian posterior values were obtained using MrBayes v.3.1.2
(Ronquist and Huelsenbeck,
2003) with the default parameters for amino acid sequences. The
same data set was also analyzed with RaxML
(Stamatakis, 2006), as
implemented on the CIPRES server
(http://www.phylo.org/)
to obtain maximum likelihood bootstrap values.
Functional expression in oocytes
RNA preparation, oocyte isolation and injection were done using standard
methods (Boland et al., 2003).
In brief, plasmids containing sponge channel cDNAs were linearized and capped
RNAs synthesized in vitro using Ambion (Austin, TX, USA) mMessage
Machine RNA polymerase kits. RNA was purified using the RNAid kit (Bio 101,
Vista, CA, USA) and concentrations were determined by spectrophotometry.
Oocytes were surgically harvested from female Xenopus laevis (Xenopus
I, Dexter, MI, USA) frogs after anesthesia by immersion in buffered 0.1%
3-aminobenzoic acid ethyl ester (Sigma Chemical Co., St Louis, MO, USA).
Oocytes were released and defolliculated by gentle agitation for 1 h or less
in 0.5 mg ml–1 collagenase A (Sigma Chemical Co.) dissolved
in a Ca2+-free solution containing (in mmol l–1):
96 NaCl, 2 KCl, 1 MgCl2, 5 Hepes, pH 7.4 with NaOH. Oocytes were
then washed and stage V/VI oocytes were injected with 50 nl of cRNA dissolved
in DEPC-treated water (5–45 ng cRNA/oocyte). Oocytes were maintained at
19°C in a frog Ringer solution of (in mmol l–1): 96 NaCl,
1 KCl, 1 CaCl2, 2 MgCl2, 10 Hepes, 5 sucrose and 2 Na
pyruvate, pH 7.4 with NaOH with 50 µ ml–1 penicillin G and
50 µgml–1 streptomycin. Electrophysiological recordings
were done 1–6 days post-injection.
Electrophysiology
K+ currents were recorded from oocytes using standard methods of
two-electrode voltage clamp (Boland et al.,
2003). We used a Geneclamp 500B amplifier (Axon Instruments,
Foster City, CA, USA) and an OC-725C amplifier (Warner Instruments, Hamden,
CT, USA). Voltage-measuring and current-passing electrodes were backfilled
with 3 mol l–1 KCl and had resistances between 0.3–1.0
M
. Currents were sampled at 5–10 kHz and filtered at 1–2
kHz. Recordings were done at room temperature (about 23°C) and oocytes
were perfused continuously during recordings. Solution composition varied by
experiment. The standard, low-chloride, external solution contained (in mmol
l–1): 5 K+ methanesulfonate (MES), 95
NMDG–MES, 2 CaCl2, 2 MgCl2, 10 Hepes, pH 7.3 with
methanesulfonic acid. K+ concentrations were adjusted by changing
the ratios of K+ and NMDG-containing solutions using either the MES
or chloride salt of K+. Other solutions are noted in figure
legends. Data were recorded on Pentium computers equipped with Digidata 1320A
(Axon Instruments) A/D hardware. Axon's Clampex aquisition and Clampfit
analysis software (v. 9) were used. Data were also transferred to Microsoft
Excel and Microcal Origin (Northampton, MA, USA) for additional analysis,
curve-fitting and the production of figures. Results were calculated from
cells with negligible background currents and only for stable cells with
membrane potentials not more depolarized than –50 mV in 5 mmol
l–1 external K+.
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|
RESULTS AND DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONReferences |
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;
Krapivinsky et al., 1998). The
channel core (M1, P and M2) had higher amino acid identity with mammalian
(38–51%) and cnidarian (48–54%) inward-rectifiers
(Table 1; supplementary
material Fig. S1).
|
The phylogenetic relationships between sponge and other metazoan inward-rectifiers are shown in Fig. 2. The overall tree topology shows that Kir diversification occurred independently in sponges, cnidarians, invertebrates and chordates and indicates that a single ancestral gene gave rise to inward-rectifiers in the metazoa.
|
;
Collins, 1998;
Sperling et al., 2007).
Computer-predicted inward-rectifier sequences from the choanoflagellate
Monosiga brevicolis (gi167515450 and gi167522960) were highly
divergent and their inclusion in the analysis destabilized all branches,
suggesting that protozoan and metazoan Kir sequences are distinct. Lack of
available sequences for ctenophores prevented a test of the recent hypothesis
that this group is the sister to other metazoans
(Dunn et al., 2008).
The order of branching of vertebrate Kir subfamilies is consistent with the
analysis by Tanaka-Kunishima and colleagues
(Tanaka-Kunishima et al.,
2007). Branching of vertebrate Kir6 after the triploblastic
invertebrates (Fig. 2) was also
not supported in their analysis. As shown previously for other ion channels
(Okamura et al., 2005), we
find that diversification of Kir channel subfamilies occurred in vertebrates
and that urochortate Kirs lie at the base of this diversification. (A
compressed archive of files used to analyze the phylogeny is available from
the authors.)
Electrophysiological properties
To study the functional characteristics of AmqKirA and AmqKirB channels, we
cloned full-length cDNAs into the pXT7 expression plasmid, prepared cRNAs by
in vitro transcription and injected purified cRNAs into frog oocytes.
From a holding potential of –50 or 0 mV, voltage steps or a ramp
protocol were used to change the membrane potential. Oocytes injected with
either AmqKirA or AmqKirB cRNAs demonstrated prominent inwardly rectifying
currents, which did not inactivate during 2 s voltage pulses. Because we
injected oocytes with only a single cRNA species, it is clear that AmqKir
channels can exist as homo-multimers. Whether they naturally co-assemble with
other Kir subunits is not yet known.
|
). These are largely
conserved in AmqKirA and AmqKirB (supplementary material Fig. S1).
|
; Scherer et al.,
2007; Zitron et al.,
2004).
Selectivity for K+
Increasing external K+ concentration,
[K+]o, caused the reversal potentials for AmqKir
currents to shift in accordance with the EK. The current
amplitude also increased in a concentration-dependent manner
(Fig. 3C). The relationship
between reversal potential and [K+]o was linear; the
fitted slope was 49 mV, close to the theoretical value of 58 for a perfectly
K+ selective channel (Fig.
3C, inset). Selectivity was further tested by perfusing with 5
mmol l–1 K+ or solutions containing 100 mmol
l–1 Na+ or 100 mmol l–1 NMDG in
the absence of K+ (Fig.
3D). Currents were negligible in the Na+ or
NMDG-containing solutions. This high K+ selectivity is typical of
most K+ channels (Hille,
2001). In addition, Na+ (0.03–1 mmol
l–1) was ineffective as a blocker of K+ (5 mmol
l–1) currents (Fig.
3D, inset).
Block by external Cs+ and Ba2+
To further characterize the electrophysiology of AmqKir channels, we tested
the effect of Cs+ and Ba2+ (monovalent and divalent
cations that block K+ current in other inward-rectifiers). Both
AmqKirA and AmqKirB currents were blocked by Cs+ and
Ba2+ (Fig. 4) but we
analyzed data for AmqKirA only because continual current run-up
(Fig. 3B) precluded accurate
measurement of the percentage block in AmqKirB. Oocytes expressing AmqKirA
were perfused with solution containing 0.03–3 mmol l–1
Cs+ (Fig. 4A) and
0.01–1 mmol l–1 Ba2+
(Fig. 4B), and current
inhibition was assessed using voltage step and ramp protocols. Block by
Cs+ showed prominent voltage-dependence in that hyperpolarization
intensified the block and depolarization relieved it. Block of AmqKirA
currents in 5 mmol l–1 K+ was
concentration-dependent with an IC50 of
37µmoll–1 for Ba2+ and
173µmoll–1 for Cs+ when measured at –140
mV (Fig. 4C).
Test of block by tertiapin-Q
Tertiapin-Q (TPN-Q), a non-oxidizable form of honeybee toxin, has been
shown to selectively inhibit a subset of Kir channels and is one of only a few
blockers available to discriminate between the contributions of different Kir
channels in situ (Ramu et al.,
2004). We tested the ability of TPN-Q to block AmqKirA, relative
to the toxin-sensitive rat Kir1.1b and toxin-insensitive mouse Kir2.1. Because
the block of Kir1.1b by TPN-Q is pH-sensitive
(Ramu et al., 2004), we
performed these experiments at an external pH of 7.6. In our experiments,
AmqKirA was insensitive to 100 nmol l–1 TPN-Q
(Fig. 4D) (N=3),
similar to mouse Kir2.1 (Fig.
4E) (N=1). Rat Kir1.1b currents, however, were blocked in
a concentration-dependent manner, with 80% block at 100 nmol
l–1 (Fig. 4F)
(N=2). The structural basis of TPN-Q selectivity is a variable region
between M1 and M2. It has been suggested that the variable region in Kir1.1
(11 residues) and Kir3.4 (10 residues) forms an alpha helix that interacts
with the helical portion of TPN-Q. Insensitivity of Kir2.1 channels may be
explained by a shorter variable region (eight residues) as a complete helical
turn may not form (Jin and Lu,
1999; Ramu et al.,
2004). The corresponding region in AmqKir channels has only four
residues, suggesting that is too short to confer sensitivity.
Concluding remarks
The electrophysiological properties of sponge Kir channels including the
absence of substantial outward current at membrane potentials positive to
EK, the high selectivity for K+ and the
voltage-dependent block by Ba2+ and Cs+ indicate that
these key features of strong inwardly rectifying K+ channels are
conserved throughout evolution. In mammals, strongly rectifying K+
channels such as Kir2 channels are typically found in skeletal muscle, cardiac
muscle and neurons (Kubo et al.,
2005; Oliver et al.,
2000). Kir2 channels function in setting the excitation threshold
by permitting membrane depolarization without much K+ efflux. Cells
expressing channels such as the AmqKir channels should be able to maintain a
stable resting potential and be able to sustain prolonged depolarizations
without massive loss of internal K+. The strong rectification of
AmqKir channels is consistent with the idea that some sponge cell membranes
may be specialized for active signaling.
The essential structural regions that control K+ selectivity have been conserved throughout the metazoa for more than 600 million years of evolution. Differences in the structures of the AmqKir channel and other metazoan Kir channels may impart functional differences not yet identified but clearly do not impact the fundamental properties of ion selectivity, block and rectification. Our results provide insight into the functional properties of the first ion channels cloned from the oldest lineage of metazoans and they shed light on properties that must have been present in the early evolution of multicellular organisms.
LIST OF ABBREVIATIONS
|
Footnotes |
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The authors thank Kenneth Kosik (University of California-Santa Barbara) for sharing sponge ion channel sequences at an early stage in this project. We are also grateful to Henry Sackin (The Chicago Medical School, Rosalind Franklin University) for the rKir1.1b cDNA, and Paulo Kofuji (University of Minnesota) for the mKir2.1 cDNA. We thank Zhe Lu (University of Pennsylvania) for a gift of recombinant tertiapin-Q. We thank Lori Spicer, Michelle Drzewiecki, and Malcolm Hill (University of Richmond) for their respective help with RNA preparation and injection, oocyte preparation and interpretation of the phylogenetic analysis. We acknowledge the contribution of The US Department of Energy Joint Genome Institute in the production of Amphimedon genomic and EST sequences. Supported by NSERC Discovery grants to W.J.G., A. N. Spencer and S.P.L., Australian Research Council grants to B.D. and by funds from the University of Richmond School of Arts and Sciences to L.M.B. Data deposition footnote: sequence data for AmqKirA and AmqKirB has been deposited to GENBANK (Accession Numbers FJ375323 and FJ375324). Conflict of Interest Statement: No conflicts declared.
* Present address: Department of Research Informatics, Amgen, Thousand Oaks,
CA 91320, USA 
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) phosphorylation sites were predicted by two out of three
prediction programs used (KinasePhos, PredPhospho and Group-based
Phosphorylation Scoring).
