From the § Department of Physiology and Biophysics,
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson
Medical School, and the Department of Molecular Biology
and Biochemistry, Rutgers, The State University of New Jersey,
Piscataway, New Jersey 08854
Received for publication, December 16, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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MinK-related peptides (MiRPs) are single
transmembrane proteins that associate with mammalian voltage-gated
K+ subunits. Here we report the cloning and
functional characterization of a MiRP MinK-related peptides (MiRPs or
KCNEs)1 are
single-transmembrane proteins that associate with pore-forming
ion-channel subunits to form stable complexes with channel properties
markedly distinct from those of the isolated pore-forming subunits (1,
2). MiRPs were identified recently in an attempt to find a To date MiRPs have been reported only in vertebrates, suggesting that
MiRPs might be relatively young in the evolutionary scale. We
speculated that they might underlie a more general role, and therefore
we sought to identify potential MiRPs homologous in lower organisms
such as the nematode Caenorhabditis elegans. The comparative
simplicity of C. elegans invites a comprehensive description
of any biological aspect of a gene. This is particularly relevant to
studies involving genetic, physiological, and structural aspects of ion
channels proteins. Thus, the existence of C. elegans MiRPs
would be of considerable interest with respect to conservation of
K+ channel subunits and to the possibility of using
C. elegans as a model system to understand how MiRPs
contribute to cellular function and how defects in these proteins might
alter cellular signaling.
We report here the cloning and functional characterization of the first
MiRP from the nematode C. elegans (mps-1).
mps-1 is expressed in the C. elegans nervous
system and represents the first example of MiRP that is essential to
neuronal excitability.
We also report the cloning and characterization of a C. elegans voltage-gated K+ channel (kvs-1)
partner for MPS-1. KVS-1 shares a significant homology with human
KV4.2, a neuronal subunit and well-recognized partner of
MiRP1 (15). In this report we show that KVS-1 is abundantly expressed
in the C. elegans nervous system and that it associates with
MPS-1 in a subset of sensory cells where both are required for normal
neuronal function.
Cloning of kvs-1 and mps-1--
Cloning of kvs-1 and
mps-1 was performed with a Smart Race kit
(Clontech) using poly(A)+ mRNA
extracted from total C. elegans RNA with a Oligotex kit (Qiagen). The primer for kvs-1 5'-RACE was
GGGCCACCAGTTCTTCTAGTGAAGGACTCA and that for 3'-RACE was
GACCGCTTGATGTATGCCCCACAGAATTTGCCACCGAACTC. The primer for
mps-1 5'-RACE was CGTACCATTAGGAGAATAATGACAAATGAGAAC and that
for 3'-RACE was GAGAGTACGAAAATGAGTTCGGTCGAATAC. cDNA was amplified
by PCR and inserted in pCI-neo vector (Promega) for functional
expression in CHO cells. All sequences were confirmed by automated DNA
sequencing. Transcripts were quantified with spectroscopy and
compared with control samples separated by agarose gel electrophoresis
stained with ethidium bromide. These novel genes have been
assigned the GenBankTM accession numbers AF541979 for
kvs-1 and AF541978 for mps-1 by the Genome Data
Base Nomenclature Committee.
Construction of Tagged Reporter Fusions to GFP--
We
constructed both translational and transcriptional GFP reporters. To
obtain transgenic nematodes expressing GFP-tagged kvs-1 and
mps-1 (translational) we employed a method developed by Yuan
et al. (16). The last ~1 kb of kvs-1 and
mps-1 were amplified by PCR from genomic DNA and joined
in-frame to the GFP reporter gene in the pPD 95.75 vector (1995 Fire
Vector Kit, generously provided by Dr. A. Fire). The reporter
constructs and the cosmid were linearized and co-injected into the
syncytial gonad of adult hermaphrodite N2 nematodes. Because the
constructs intentionally lack the promoter and the initial methionine,
they are not translated without recombination with the cosmid (which
contains the entire gene and its promoter). Thus, this strategy
increases the likelihood of detecting all tissue-specific expression,
because all upstream regulatory sequences are incorporated, and it
ensures high specificity, because it requires the cosmid for
recombination. For kvs-1::gfp the primers were (5' direction) ATGGATCCGATGACAACTGTTGGATATGGAGA and
CCGGTACCGTTTCTGCCACATCAATAGGTCGTTG in the forward and reverse direction, respectively. For mps-1::gfp
the primers were ATGGATCCTATTAAGCTACATGAAGTTCTCTA and
ACGGTACCGTCACGTCTAAGCTAAATGATTTATC, respectively. For transcriptional reporters, because kvs-1 possesses a large (~10 kb) first
intron (see Fig. 1D), we constructed two reporter
constructs, Pkvs-1::gfp1 and
Pkvs-1::gfp2, corresponding to
genomic sequence preceding exons 1 and 2, respectively. To create
Pkvs-1::gfp1, a ~3-kb genomic
fragment upstream of the kvs-1 gene containing 15 bp of the
open reading frame was amplified by PCR and subcloned into pPD95.75.
Primers were: ATCTAGTCTAGAGATTACGAACGTATTTTCACGAGG and
AGTCGCGGATCCACATCAGCCTTTCCGTGCTCATGT in the forward and reverse direction, respectively. To construct
Pkvs-1::gfp2, a ~4-kb
fragment of intronic sequence upstream exon 2 containing 20 bp of the
exon was cloned into pPD95.75. Primers were:
GCTACTTCTAGAAGTGACAGTCTTGAGGCAAAATC and
AGTAGCGGATCCGGCAATTTGGTTGCTGTCTTTGTCAC. Both
reporters gave rise to GFP expression.
Pmps-1::gfp was similarly
constructed by amplification and cloning into pPD95.75 of a ~2-kb
fragment upstream exon 1. Primers were
CGAATGTGGCTGCTCAATCGAGG and
TTAGAGTACTGTCAGTCAGAGTTAATA. Worms were analyzed and
photographed with a Zeiss Axioplan 2 microscope equipped with a digital camera.
Germline Transformation--
Plasmids were microinjected at the
concentration of 25 ng/µl. The transformation marker was
lin-15(+) (50 ng/µl). Five transgenic lines carrying
extrachromosomal arrays were identified for each construct.
Dye Filling Experiments--
Transgenic worms were picked to a
plate containing DiD (Molecular Probes) diluted in M9 buffer (0.01 mg/ml) and allowed to stain for 2-3 h at room temperature. Worms were
then transferred to an agar plate and allowed to crawl on the bacterial
lawn for about 15 min to destain.
Phenotypic Analysis--
For dsRNA production, ~ 0.7-kb
regions of kvs-1 and mps-1 genes, designed to
minimize identity (<30%) with other C. elegans genes were
amplified by PCR with oligonucleotides that added 5' T7 promoter
sequence. For kvs-1 the primers were
5'-TAATTAATACGACTCACTATAGGGGTGATTCCCAGCACCAGGAAAGGAC and
5'-TAATTAATACGACTCACTATAGGGAACCAGTCCAATAACACTAATAAGAACAAAAG, and
cDNA was used as the template. For mps-1 the primers
were 5'-TAATTAATACGACTCACTATAGGGCGAGAAGAATTAGAAATACAGGAACG and
5'-TAATTAATACGACTCACTATAGGGATGAAGATGACGAAGATGACATGGG, and
template was genomic DNA. dsRNAs in vitro synthesis was with a MEGAscript kit (Ambion) using the PCR products as templates. The
reactions were annealed at 37 °C for 30 min after denaturation at
68 °C for 10 min. RNA was analyzed by agarose gel electrophoresis to
verify that it was double-stranded. Approximately 100 pl of dsRNA (1.5 µg/µl in H2O) was injected into both gonads of young adults. Worms were allowed to lay the eggs contained in the uterus for
2-3 h and then transferred separately onto fresh plates. F1 progeny of
injected worms was analyzed. To assay thrashing (17), L4 worms
were picked in a drop of M9 buffer on an agar plate. After 2 min of
recovery, thrashes were counted for 2 min. A thrash was defined as a
change in the body bend at the mid-body point. Tests for response to
light touch (18) were performed without knowledge of the genotypes of
the worms. Single worms were picked to individual plates and tested six
times for response to light touch to the head and tail with an eyelash.
Responses to head/tail touch were recorded as backward/forward
movement. The overall response to touch of each group of worms was
expressed as the average percentage of times the worms responded. In
osmotic avoidance assays (19), animals were placed on an agar plate
within a 1-cm-high osmotic strength ring consisting of 8 M
glycerol. Assays were scored after 8 min for retention or escape from
the ring. For the volatile attractants assays, 1 µl of 20 mM NaN3 was applied to the centers of two tests
spots 15 min prior to the experiment on opposite sides of a plate.
Twenty worms were placed at the center of the plate between the two
spots, 1 µl of the test odorant was placed at one spot, and 1 µl of
ethanol was applied to the other spot. For water-soluble attractants
(20), a chunk of agar 0.5-cm in diameter was removed from 10-cm plates
and soaked in the attractant for 2 h. Chunks were put back in the
plate overnight to allow equilibration and formation of a gradient.
Twenty worms were placed between this spot and a control spot on the
opposite side of the plate. Ten microliters of 20 mM
NaN3 was placed on both spots. Lysine and biotin were used
at concentrations of 0.5 and 0.2 M, respectively,
benzaldehyde at a 1:200 dilution, and isoamyl alcohol at a 1:100
dilution (21). After 1 h of chemotaxis, animals on the two
different sides of the plate (the one with the attractant and the one
with the control) were counted, and a C.I. was calculated as the number
of animals at the test spot minus the number of animals at the control
spot, divided by the total number of animals on the plate. A positive
C.I. indicated an attraction to the odor. For collision nose-touch
assays (19), worms were tested 10 times each with an eyelash placed in
front of them on the agar plate. Avoidance was quantitated as the
percentage of trials in which animals responded to touch by stopping
forward movement or reversing.
Immunocytochemistry--
A c-Myc epitope was fused to the N
terminus of MPS-1 by PCR. Transiently transfected cells were incubated
in fresh complete media containing the monoclonal anti-c-Myc antibody
(40 µg/ml, clone 9E10; Roche Molecular Biochemicals) at 37 °C for
1 h. Cells were washed once with phosphate-buffered saline (PBS)
and fixed with paraformaldehyde (4% in PBS) for 15 min at room
temperature. After fixing, cells were washed three times for 5 min with
PBS and blocked for 1 h at room temperature with 5% nonfat dry
milk in PBS plus 0.1% Tween 20. Cells were incubated with the
secondary antibody, Cy3-conjugated goat anti-mouse (Jackson
ImmunoResearch) (1:2000, in 5% nonfat dry milk in PBS plus 0.1% Tween
20), for 1 h at room temperature, and subsequently washed three
times for 5 min with PBS.
Electrophysiology--
CHO cells were transiently transfected
with cDNA ligated into pCI-neo using a SuperFect kit (Qiagen) and
studied after 24-36 h. Data were recorded with an Axopatch 200B
(Axon), a PC (Dell), and Clampex software (Axon), filtered at 1 kHz,
and sampled at 2.5 kHz. Bath solution was (in mM): 4 KCl,
100 NaCl, 10 Hepes (pH 7.5 with NaOH), 1.8 CaCl, and 1.0 MgCl. Pipette
solution: 100 KCl, 10 Hepes (pH 7.5 with KOH), 1.0 MgCl, 1.0 CaCl, and
10 EGTA (pH 7.5 with KOH).
Using in silico approaches, we identified a predicted
MiRP-related C. elegans protein, C29F5.4 (mps-1)
and confirmed its expression and primary sequence by analysis of
reverse transcription-PCR products (Fig.
1A-C). Interestingly, the
MPS-1 N terminus and transmembrane domain exhibit significant homology
to human MiRP1, but the C terminus is more similar to human MiRP3,
suggesting that MiRP1 and MiRP3 might be derived from a common ancestor
shared with nematodes (Fig. 1B).
-subunit, MPS-1, and of a
voltage-gated pore-forming potassium subunit, KVS-1, from the nematode
Caenorhabditis elegans. mps-1 is expressed in
chemosensory and mechanosensory neurons and co-localizes with
kvs-1 in a subset of these. Inactivation of either
mps-1 or kvs-1 by RNA interference (RNAi)
causes partially overlapping neuronal defects and results in
broad-spectrum neuronal dysfunction, including defective chemotaxis,
disrupted mechanotransduction, and impaired locomotion. Inactivation of
one subunit by RNAi dramatically suppresses the expression of the
partner subunit only in cells where the two proteins co-localize.
Co-expression of MPS-1 and KVS-1 in mammalian cells gives rise to a
potassium current distinct from the KVS-1 current. Taken together these
data indicate that potassium currents constitute a basic determinant
for C. elegans neuronal function and unravel a unifying
principle of evolutionary significance: that potassium channels in
various organisms use MiRPs to generate uniqueness of function with
rich variation in the details.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit for the cardiac potassium channel HERG, which in heterologous expression systems behaves differently than in native cardiomyocytes. Using in silico approaches three MiRPs (MiRP1, MiRP2, and
MiRP3) were recognized by their homology with MinK, a putative
-subunit of HERG and KCNQ1 (2-5). The last member of the family,
MiRP4 was identified later (6). Although MiRPs were initially
identified as cardiac proteins, they were soon found to be expressed
and function in other tissues and to cause acquired and congenital disease (2, 7-11). For instance, MiRP2 is expressed in skeletal muscle
where it associates with Kv3.4 and, when defective, can cause periodic
paralysis (8). Mutations in MinK and MiRP1 genes can lead to Long QT
syndrome, a specific form of polymorphic ventricular tachycardia
characterized by impaired ventricular repolarization (2, 10-12). A
well-established characteristic of MiRPs is the capacity to associate
with multiple pore-forming subunits in heterologous systems. For
instance, MiRP1 can associate with HERG, KCNQ1, HCN, and Kv4.2 subunits
(2, 13-15). Indeed, MiRP "promiscuity" has considerable biomedical
implications, because, if a single MiRP co-assembles with multiple
pore-forming subunits, genetic mutations would be predicted to lead to
disruption of multiple currents simultaneously.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The C. elegans genome
encodes a MiRP-related protein and a Kv4.2-related protein that is a
candidate interaction partner. A, the genomic
organization of mps-1. Exons are indicated by
boxes, introns by lines. The gene is spliced in
six exons in contrast to human MiRPs that are generally contained in a
single exon. B, MPS-1 protein sequence and alignments with
MiRP1 (20% identity and 59% homology) and MiRP3 (20% identity and
61% homology). Alignment by ClustalW (available at bioweb.pasteur.fr).
C, hydropathy plot of MPS-1. Plot was calculated with the
Kyte and Doolittle algorithm (available at
bioweb.pasteur.fr/seqanal/interfaces/toppred.html). Top, a
schematic representing MPS-1 predicted topology. The
secondary structure predicts a single-span protein that has the
basic topology of mammalian MiRPs and that is an extracellular N
terminus, a single transmembrane domain, and an intracellular C
terminus. The branch structure indicates a potential
N-glycosylation site, a feature common to other members of
the family. D, the genomic organization of kvs-1.
Roughly 10% of the genomic sequence encodes the cDNA.
E, the protein sequence of KVS-1 and alignment with the
human voltage-gated KV4.2 (20% identity and 40%
homology). Transmembrane helices (S1-S6) and the
potassium-channel signature are indicated. F, hydropathy
plot of KVS-1. Top, the predicted topology of KVS-1
forecasts an ion-channel protein with a large intracellular N domain,
six transmembrane domains, including an S4 span with multiple
positively charged residues arrayed as in other channels activated by
voltage and a pore loop with a potassium channel signature
sequence.
MiRPs do not form channels on their own (2). The C. elegans genome encodes many potassium channel homologues that could be potential interaction partners of MPS-1 (22). We focused on predicted potassium channel subunit C53C9.3 (kvs-1) (Fig. 1, D-F) as a candidate partner for MPS-1, because 1) potassium channel C53C9.3/KVS-1 shares significant homology with human KV4.2, known to associate with MiRP1 (15) and 2) microarray data suggest mps-1 and kvs-1 gene expression is co-regulated in different growth conditions and at different developmental stages (23).
We determined expression patterns of mps-1 and
kvs-1 by analyzing transgenic animals harboring
translational and transcriptional reporter fusions.
We found mps-1 expression in
ASG, ASE, ADF, and ASH neurons (Fig. 2, A-F, and Table
I; we confirmed identities of the latter
by scoring for DiD filling (24)), AWC (see Fig. 4A and Table
I), in ALM and PLM touch-sensing neurons (Fig. 2, G and
H), and in the vulva (Fig. 4B). We detected
kvs-1 expression in more than 10 cells in the head,
including the amphid neurons ADL, ASK, ASH, ADF, ASE, AWC (Fig.
2, I-Q, and Table I), and ASG (not shown, Table I),
in ventral cord neurons (Fig. 2R), in the
motoneuron PDA (not shown) in the anal depressor muscle (Fig.
2S) and in sperm (not shown). Thus, MPS-1 has the potential to influence the activities of multiple potassium channels expressed in
diverse neurons and cell types. Moreover, the co-expression of
mps-1 and kvs-1 in AWC, ADF, ASG, ASE, and ASH
neurons suggests MPS-1 and KVS-1 may form a functional complex in these
amphid neurons, which mediate diverse chemosensory responses
(25).
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To probe the physiological roles of kvs-1 and
mps-1 in nervous system, we screened for potential
behavioral defects predicted for impaired neuronal function of the
cells that appear to express kvs-1 and mps-1
using double-stranded RNA-mediated gene inactivation (RNAi). RNAi was
fairly effective in knocking down the expression of GFP-tagged MPS-1
and KVS-1 proteins (Figs. 4, C-D and K-L) confirming RNAi targeting of their transcripts. mps-1 RNAi
induced defects in body touch sensation (Fig.
3A), chemotaxis to biotin and
lysine, osmotic avoidance, and nose-touch collision (Fig. 3,
B-E), phenotypes that correlate well with the detected
presence of mps-1 in ALM and PLM touch-sensing neurons (body
touch sensation) and in ASG, ADF, and ASE (chemotaxis to biotin and
lysine) and ASH amphid neurons (osmotic avoidance and nose-touch, see
Fig. 3 legend and Table I). mps-1 RNAi did not affect
chemotaxis to benzaldehyde (Fig. 3G) and isoamyl alcohol
(Fig. 3H) that are mediated by AWC neurons where
mps-1 appears to be expressed, and, as expected, it did not
affect other sensory functions such as chemotaxis to diacetyl, octanol
avoidance, and thermotaxis that are mediated by neurons that not appear
to express mps-1. We conclude that mps-1 is
required for the normal function of several neuronal types in which
this MiRP is expressed.
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Importantly, like mps-1 RNAi-treated animals, kvs-1 RNAi-treated animals exhibited impaired chemotaxis to lysine and to biotin (Fig. 3, B and C), defective osmotic avoidance (Fig. 3D), and nose-touch responses (Fig. 3E), consistent with compromised ADF, ASG, ASE, and ASH function (Fig. 3 and Table I) (19, 20, 25, 26). Nematodes co-injected with a mixture of dsRNA of both mps-1 and kvs-1 did not exhibit significant enhancement in defects in osmotic avoidance and nose-touch (Fig. 3, D and E). This suggests phenotypes we observe reflect maximal disruption of these behaviors and that the two subunits contribute similarly to the behaviors, possibly as parts of the same channel complex.
Consistent with the kvs-1 cellular expression pattern, RNAi induced defective forward movement (Fig. 3F) and defective chemotaxis to benzaldehyde (Fig. 3G) and to isoamyl alcohol (Fig. 3H). Benzaldehyde and isoamyl alcohol chemotaxis are specifically mediated by AWC neurons, which appear to express kvs-1 (25). Other sensory functions such as thermotaxis and octanol avoidance, controlled by neurons that did not express kvs-1, were unaffected.
To test the hypothesis that MPS-1 and KVS-1 belong to the same channel
complex in some sensory neurons, we assessed the effect that epigenetic
inactivation of each gene has on expression of its putative partner.
For this purpose we exploited
kvs-1::gfp and
mps-1::gfp transgenic nematodes that
express KVS-1 and MPS-1 subunits fused to GFP proteins. Inactivation of
kvs-1 by RNAi dramatically suppressed
mps-1::gfp signals only in those cells where the two proteins co-localize, that is ADF and ASE (Fig. 4, A-F, and O),
and ASG and ASH (not shown) with the exception of AWC neurons.
Reciprocally, mps-1 RNAi selectively inhibited kvs-1::gfp fluorescence in the same
neurons (ADF and ASE (Fig. 4, I-N and P) and ASG
and ASH (data not shown)). Thus, it appears that in the cells where
both proteins co-localize the stability of KVS-1 depends on MPS-1 and
vice versa. These data are consistent with the observation
that in ASH neurons simultaneous inactivation of both genes produces
the same effect as inactivation of each gene separately (Fig. 3,
D and E). Furthermore, they may account for the
fact that mps-1 and kvs-1 RNAi impair neuronal
functions to the same extent in the neurons where they co-localize
(Fig. 3, B-E). They may also imply that, in the cells where
the two subunits do not co-localize, they might associate with other
unidentified endogenous subunits rather than exist in homomeric form.
Moreover, they suggest that MPS-1 and KVS-1 might interact to form a
channel complex early during protein biosynthesis. Alternatively, each subunit might be required to confer structural stability to the complex
at the plasma membrane, although this possibility seems unlikely,
because in mammalian expression systems, both KVS-1 (Fig.
5A) and MPS-1 (Fig. 4,
G and H) appear to be stable when expressed
alone.
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Because our data suggested that MPS-1 associates with KVS-1 in some amphid neurons, we conducted electrophysiological studies to compare the functional properties of KVS-1 channels alone and with MPS-1. We expressed KVS-1 and MPS-1 in Chinese hamster ovary (CHO) cells and used the whole-cell configuration of the patch clamp to characterize currents. When we transfected KVS-1 cDNA alone, we found a novel voltage-gated, potassium-selective ion channel opened by depolarization. The opposing effect of rapid activation and inactivation generated a typical "A-type" profile, characteristic of the human KV1 and KV4 potassium channel sub-families (Fig. 5A) (27). By progressive substitution of bath potassium with sodium, we found the KVS-1 channel to be selective for potassium, which shifted reversal potential by kT/e = 20.5 ± 1.7 mV (Fig. 5D). We assessed monovalent cation selectivity by determining bi-ionic permeability ratios and found the KVS-1 profile corresponded to a type IV series, potassium > rubidium > cesium > sodium = lithium, with sodium ~100-fold less permeant than potassium (PNa/PK ~ 0.01) (n = 7; data not shown). Thus, C. elegans KVS-1 encodes a K+-selective channel with properties similar to mammalian channels of the Kv4 family, indicating functional conservation suggested by sequence homology.
As expected from work with mammalian MiRPs, when we transfected MPS-1
cDNA into CHO cells, we failed to find any significant current
(n = 40, Fig. 5B), although
immunocytochemical assays indicated that MPS-1 subunits were present at
the plasma membrane (Fig. 4, G and H) (28).
However, when we co-transfected MPS-1 with KVS-1, we identified several
novel features of the introduced current, suggesting co-assembly (Fig.
5C). As expected, MPS-1·KVS-1 channels were selective for
potassium (kT/e = 19.0 ± 1.7 mV, Fig. 5D). Heterologously expressed macroscopic KVS-1 currents
showed half-maximal activation (V1/2) at 44.6 ± 1.8 mV with a slope factor (Vs) of 21.7 ± 1.7 mV (Fig. 5E). Co-expression with MPS-1 induced a left
shift in the midpoint for activation (V1/2 = 32.6 ± 1.2 mV) without significantly affecting the
voltage-dependence (Vs = 20.1 ± 1.1).
Although KVS-1 subunits inactivated with a rate of = 39.9 ± 4.4 ms at +120 mV, MPS-1·KVS-1 channels were faster (
= 21.3 ± 3.0 ms, Fig. 5F). We quantified recovery from
inactivation with whole-cell currents using 1.2-s and 0.2-s
depolarizing pulses at +60 mV spaced out by progressively longer
periods at
80 mV (Fig. 5G). The time course was best fit
to a single-exponential function with
= 9.1 ± 0.2 ms for
KVS-1 alone and
= 20.0 ± 0.6 ms with MPS-1·KVS-1.
MPS-1 effects extended to pharmacology. We tested tetraethylammonium
and 4-aminopyridine (4-AP), two classic potassium channel blockers that
have been assessed on ASE native potassium currents and on Kv4 family
members (29-31). We found that both cloned channels were resistant to
tetraethylammonium (20 mM). In contrast, 4-AP inhibited
MPS-1·KVS-1 complexes more efficiently than channels formed with
KVS-1 subunits alone. Dose-response curves revealed equilibrium
inhibition constants of Ki = 6.2 ± 1.0 mM for MPS-1·KVS-1 channels and Ki = 25.6 ± 4.1 mM for channels formed with KVS-1 subunits
alone. In both cases, the relationships of fractional current and 4-AP
fit well to the Hill function with n ~ 1 suggesting
that a single 4-AP molecule inhibits one channel complex (Fig.
5I). Taken together, electrophysiological and
pharmacological analyses establish that a C. elegans MiRP
can alter the properties of a specific C. elegans K+ channel, demonstrating functional conservation of MiRP
activities and suggesting that a MPS-1· KVS-1 channel may assemble
in vivo to influence neuronal function.
The human MiRP1 and Kv4.2 subunits co-assemble in heterologous
expression systems (15). Channels formed by hKv4.2 subunits, alone and
with hMiRP1, have been characterized in Xenopus oocytes (15,
31). Homomeric hKv4.2 channels inactivate and recover from inactivation
at rates ~13 and ~300 ms, respectively (at +60 mV), and are
inhibited by 4-AP in the millimolar range (~1 mM). Co-assembly with hMiRP1 slows inactivation kinetics (~44 ms), does
not affect recovery from inactivation, and increases 4-AP residency
time probably by hindering conformational changes of the channel pore
(15). The analogy of this channel complex with C. elegans
MPS-1·KVS-1 argues that some structural mechanisms by which MiRPs act
to alter channel function might be conserved among MiRPs of different
species. To test this hypothesis we expressed and studied functionally
hybrid channels formed with human MiRP1 and C. elegans KVS-1
and with C. elegans MPS-1 and human Kv4.2. Interestingly,
both MiRPs were able to alter the attributes of pore-forming subunits
distant in the evolutionary scale. Thus, like MPS-1, hMiRP1-speeded
KVS-1 inactivation kinetics ( = 17.5 versus 39.9 ms
at +120 mV, Fig. 6, A and
B) slowed down recovery from inactivation (
= 25 versus 9 ms, Fig. 6C) and increased the
sensitivity of the channel to 4-AP (Ki = 12.6 versus 25.6 mM, Fig. 6D).
Reciprocally, MPS-1 slowed down hKv4.2 inactivation kinetics (
= 24.5 versus 12.3 ms, Fig. 6, E and
F), did not alter recovery from inactivation (
= 357 versus 294 ms, Fig. 6G) and decreased 4-AP
susceptibility (Ki = 6.8 versus 1.7 mM, Fig. 6H). MiRPs did not alter the voltage
dependence of activation (not shown). We conclude that the
MiRP/K+ channel relationship might be highly conserved from
invertebrates to humans.
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KVS-1 Is a Novel K+ Subunit That Contributes to the C. elegans Neuronal Current IK-- We report here the identification of a novel C. elegans K+ channel gene, kvs-1, that is required for the normal function of several C. elegans neurons. KVS-1 is broadly expressed in nervous system, in anal depressor muscle, and in sperm. Disruption of KVS-1 function by RNAi is associated with a variety of neuronal defects ranging from impaired locomotion to defective chemotaxis. These defects correlate well with the cellular expression pattern of the KVS-1 protein. More specifically, kvs-1 RNAi leads to defective chemotaxis to lysine, biotin benzaldehyde isoamyl alcohol, osmotic avoidance and nose-touch, which are specific functions of neurons such as ASK, ASH, ASG, ASE, AWC, and ADF (25).
Unlike vertebrates, C. elegans neurons can signal effectively without classic sodium action potentials (the C. elegans genome does not encode voltage-gated sodium channel genes) (22, 30, 32). Elegant studies on ASE neurons suggest that voltage-dependent potassium currents play a fundamental role for the functions of C. elegans neurons by contributing to both the maintenance and modulation of cell sensitivity (30). Our data support this notion by the identification and functional characterization of this novel voltage-gated potassium channel.
MPS-1 Associates with KVS-1 in a Subset of Amphid Neurons-- C. elegans mps-1 is the first reported invertebrate MiRP homologue. We propose that in ADF, ASG, ASE, and ASH neurons, MPS-1 works in conjunction with specific K+ channel KVS-1 to modulate the electrical activity of these neurons. First, kvs-1 and mps-1 expression overlaps in these cells. Second mps-1 RNAi is consistent with altered function of these sensory neurons. In particular, the simultaneous inactivation of mps-1 and kvs-1 leads to effects on ASH function that are identical to those provoked by inactivation of each gene separately arguing that the two subunits could contribute to the same channel complex. Third, each subunit controls expression and/or stability of the other in neurons where both are expressed. Although the mps-1 knockdown effects on KVS-1 expression are consistent with previous observations showing that mammalian MiRPs alter protein expression levels of some pore-forming subunits (4, 14, 33), kvs-1 RNAi effect on MPS-1 protein levels is a novel observation suggesting that neither subunit is dispensable in physiological cell types in which a complex is programmed to be assembled. Fourth, in CHO cells KVS-1 forms functional channels whose characteristics are markedly altered upon co-expression with MPS-1. Thus MPS-1 alters voltage dependence of activation, inactivation, residual current, recovery from inactivation, and susceptibility to the K+ channel blocker 4-aminopyridine. Interestingly, both KVS-1 and MPS-1 in CHO cells are required to produce a current that has similar characteristics to native currents found in neurons, such as ASEs (29, 30, 32), that endogenously express mps-1 and kvs-1. Overall, several lines of evidence corroborate the notion that in some amphid neurons KVS-1 and MPS-1 associate to form a potassium channel complex that plays an important physiological role. Further efforts will be required to ascertain, however, whether additional endogenous subunits might contribute in these cells to form native MPS-1·KVS-1 complexes.
mps-1 Is a Gene Influencing Several Neuronal Functions-- MPS-1, like KVS-1, is involved in the control of a variety of neuronal functions. Some defects are common with kvs-1 phenotypes. Others, such as body touch sensation, are characteristic only of mps-1 and argue that MPS-1 might promiscuously partner with multiple pore-forming subunits. The identity of other putative partners for MPS-1 has still to be ascertained, however, the physiological relevance of MiRP promiscuity, as well the potential for leading to multiple dysfunction when mps-1 is disrupted, is now validated. We did notice one cell type in which MPS-1 and KVS-1 are co-expressed but do not appear to co-assemble, the well-characterized AWC neurons. Specific chemosensory functions mediated by AWC neurons such as chemotaxis to benzaldehyde and isoamyl alcohol (25) are unaffected by mps-1 RNAi but are disrupted by kvs-1 RNAi. Consistent with the observation, kvs-1 knockdown does not alter mps-1 expression in this cell type. At present the role of mps-1 in AWC cells as well as the identity of interaction partners remains elusive, but we note that subtle defects in adaptation or developmental specificity may have been missed in our general chemosensation assays.
MiRPs Share Common Structural Determinants across Phyla-- Our data suggest that MiRPs might play conserved roles in modulation of K+ channel function. MPS-1, which displays a high level of homology with human MiRP1, especially in the N terminus, can modulate hKv4.2 function and so too hMiRP1 modifies KVS-1 functional attributes. 4-AP binding and hKv4.2 channel inactivation are mutually exclusive, arguing that the structures that are important for inactivation might also be involved in 4-AP binding (31). Probably 4-AP binds to a site at, or adjacent to, the domains involved in channel inactivation and induces conformational changes in the channel preventing ion permeation through the pore (31). MiRPs simultaneously influence channel inactivation and 4-AP blockade, thus disclosing an intimate correspondence between the role played by MiRPs in the two channel complexes. Although it is not clear yet whether MiRPs alter susceptibility to 4-AP by acting on the binding site or by modifying the geometry of the pore, these data suggest the existence of general structural and functional principles that seem to be conserved among MiRPs of diverse species. This correspondence also validates the possibility to use in the future the C. elegans MPS-1·KVS-1 complex as a useful tool to investigate biochemical and biophysical aspects of its human homologue MiRP1·Kv4.2.
MiRPs May Provide Functional Diversity in the C. elegans Nervous
System--
Our data provide molecular evidence to the general idea
that a significant degree of heterogeneity in neuronal potassium flux underlies functional differences among neurons (30). Such complexity is
also observed in the human nervous system (34) highlighting a general
principle conserved across species. The molecular bases for neuronal
diversity may arise through differential expression of potassium
channel -subunits with further diversity accomplished by combination
of MiRPs with K+ channel
-subunits. This combinatorial
arrangement is advantageous from a genetic point of view, because it
provides multiplicity and uniqueness through combinations of only a few
gene products. In this study we found that both mps-1 and
kvs-1 are expressed in, and required for the function of,
many neuronal types. In humans MiRPs are essential to many biological
functions and can lead to inherited and/or acquired disease (2, 8, 10,
11, 35). Here we show that MiRPs are not restricted to higher
organisms; rather, MiRP modulation of channel function may represent an
ancient mechanism to achieve functional diversity in the nervous system.
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ACKNOWLEDGEMENTS |
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We are deeply indebted with Cinzia Sesti for her unconditional support during the development of this work. We thank Dr. Steve Goldstein for helpful discussion and for the Kv4.2 clone.
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FOOTNOTES |
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* This work was funded in part by National Institutes of Health Grant R01 NS 37955 (to M. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF541979 and AF541978.
¶ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-4032; Fax: 732-235-5038; E-mail: sestife@umdnj.edu.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M212788200
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ABBREVIATIONS |
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The abbreviations used are: MiRP, MinK-related peptide; C.I., chemotaxis index; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; GFP, green fluorescence protein; dsRNA, double-stranded RNA; PBS, phosphate-buffered saline; RNAi, RNA interference; 4-AP, 4-aminopyridine.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Abbott, G., and Goldstein, S. (1998) Q. Rev. Biophys. 31, 357-398[CrossRef][Medline] [Order article via Infotrieve] |
2. | Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., and Goldstein, S. A. (1999) Cell 97, 175-187[Medline] [Order article via Infotrieve] |
3. | Barhanin, J., Lesage, F., Guillemare, E., Fink, M., Lazdunski, M., and Romey, G. (1996) Nature 384, 78-80[CrossRef][Medline] [Order article via Infotrieve] |
4. | McDonald, T. V., Yu, Z., Ming, Z., Palma, E., Meyers, M. B., Wang, K. W., Goldstein, S. A., and Fishman, G. I. (1997) Nature 388, 289-292[CrossRef][Medline] [Order article via Infotrieve] |
5. | Finley, M. R., Li, Y., Hua, F., Lillich, J., Mitchell, K. E., Ganta, S., Gilmour, R. F., Jr., and Freeman, L. C. (2002) Am. J. Physiol. 283, H126-H138 |
6. | Piccini, M., Vitelli, F., Seri, M., Galietta, L. J., Moran, O., Bulfone, A., Banfi, S., Pober, B., and Renieri, A. (1999) Genomics 60, 251-257[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Cowley, E. A.,
and Linsdell, P.
(2002)
J. Physiol.
538,
747-757 |
8. | Abbott, G. W., Butler, M. H., Bendahhou, S., Dalakas, M. C., Ptacek, L. J., and Goldstein, S. A. (2001) Cell 104, 217-231[Medline] [Order article via Infotrieve] |
9. | Grahammer, F., Herling, A. W., Lang, H. J., Schmitt-Graff, A., Wittekindt, O. H., Nitschke, R., Bleich, M., Barhanin, J., and Warth, R. (2001) Gastroenterology 120, 1363-1371[Medline] [Order article via Infotrieve] |
10. |
Sesti, F.,
Abbott, G. W.,
Wei, J.,
Murray, K. T.,
Saksena, S.,
Schwartz, P. J.,
Priori, S. G.,
Roden, D. M.,
George, A. L., Jr.,
and Goldstein, S. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
10613-10618 |
11. |
Cui, J.,
Kagan, A.,
Qin, D.,
Mathew, J.,
Melman, Y. F.,
and McDonald, T. V.
(2001)
J. Biol. Chem
276,
17244-17251 |
12. | Splawski, I., Tristani-Firouzi, M., Lehmann, M. H., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340[Medline] [Order article via Infotrieve] |
13. |
Tinel, N.,
Diochot, S.,
Borsotto, M.,
Lazdunski, M.,
and Barhanin, J.
(2000)
EMBO J
19,
6326-6330 |
14. | Yu, H., Wu, J., Potapova, I., Wymore, R. T., Holmes, B., Zuckerman, J., Pan, Z., Wang, H., Shi, W., Robinson, R. B., El-Maghrabi, M. R., Benjamin, W., Dixon, J., McKinnon, D., Cohen, I. S., and Wymore, R. (2001) Circ. Res. 88, E84-E87[Medline] [Order article via Infotrieve] |
15. |
Zhang, M.,
Jiang, M.,
and Tseng, G.
(2001)
Circ. Res.
88,
1012-1019 |
16. | Yuan, A., Dourado, M., Butler, A., Walton, N., Wei, A., and Salkoff, L. (2000) Nat. Neurosci. 3, 771-779[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Miller, K. G.,
Alfonso, A.,
Nguyen, M.,
Crowell, J. A.,
Johnson, C. D.,
and Rand, J. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12593-12598 |
18. | Driscoll, M., and Chalfie, M. (1991) Nature 349, 588-593[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Hart, A. C.,
Kass, J.,
Shapiro, J. E.,
and Kaplan, J. M.
(1999)
J. Neurosci.
19,
1952-1958 |
20. | Bargmann, C. I., and Horvitz, H. R. (1991) Neuron 7, 729-742[Medline] [Order article via Infotrieve] |
21. | Bargmann, C. I., Hartwieg, E., and Horvitz, H. R. (1993) Cell 74, 515-527[Medline] [Order article via Infotrieve] |
22. |
Bargmann, C.
(1998)
Science
282,
2028-2033 |
23. |
Kim, S. K.,
Lund, J.,
Kiraly, M.,
Duke, K.,
Jiang, M.,
Stuart, J. M.,
Eizinger, A.,
Wylie, B. N.,
and Davidson, G. S.
(2001)
Science
293,
2087-2092 |
24. | Perkins, L. A., Hedgecock, E. M., Thomson, J. N., and Culotti, J. G. (1986) Dev. Biol. 117, 456-487[Medline] [Order article via Infotrieve] |
25. | Bargmann, C., and Mori, I. (1997) in C. elegans II (Riddle, D. L. , Blumenthal, T. , Meyer, B. J. , and Priess, J. R., eds) , pp. 717-737, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
26. | Kaplan, J., and Horvitz, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2227-2231[Abstract] |
27. | Hille, B. (2001) Ionic Channels of Excitable Membranes , 3rd. Ed. , p. 138, Sinauer Associates, Sunderland, MA |
28. |
Bianchi, L.,
Shen, Z.,
Dennis, A. T.,
Priori, S. G.,
Napolitano, C.,
Ronchetti, E.,
Bryskin, R.,
Schwartz, P. J.,
and Brown, A. M.
(1999)
Hum. Mol. Genet.
8,
1499-1507 |
29. | Christensen, M., Estevez, A., Yin, X., Fox, R., Morrison, R., McDonnell, M., Gleason, C., Miller, D. M., 3rd, and Strange, K. (2002) Neuron 33, 503-514[Medline] [Order article via Infotrieve] |
30. | Goodman, M. B., Hall, D. H., Avery, L., and Lockery, S. R. (1998) Neuron 20, 763-772[Medline] [Order article via Infotrieve] |
31. | Tseng, G., Jiang, M., and Yao, J. (1996) J. Pharmacol. Exp. Ther. 279, 865-876[Abstract] |
32. | Pierce-Shimomura, J. T., Faumont, S., Gaston, M. R., Pearson, B. J., and Lockery, S. R. (2001) Nature 410, 694-698[CrossRef][Medline] [Order article via Infotrieve] |
33. | Schroeder, B. C., Waldegger, S., Fehr, S., Bleich, M., Warth, R., Greger, R., and Jentsch, T. J. (2000) Nature 403, 196-199[CrossRef][Medline] [Order article via Infotrieve] |
34. | Song, W. (2002) Neurosci. Res. 42, 7-14[CrossRef][Medline] [Order article via Infotrieve] |
35. | Isbrandt, D., Friederich, P., Solth, A., Haverkamp, W., Ebneth, A., Borggrefe, M., Funke, H., Sauter, K., Breithardt, G., Pongs, O., and Schulze-Bahr, E. (2002) J. Mol. Med. 80, 524-532[CrossRef][Medline] [Order article via Infotrieve] |
36. | Thomas, J. H., and Lockery, S. (1999) in C. elegans (Hope, I. A., ed) , pp. 143-179, Oxford University Press, Oxford, UK |