Mutations in Mcoln3 associated with deafness and
pigmentation defects in varitint-waddler (Va) mice
Federica Di Palma*, Inna A. Belyantseva†, Hung J. Kim*‡, Thomas F. Vogt§¶, Bechara Kachar†,
and Konrad Noben-Trauth*?
*Section on Neurogenetics, Laboratory of Molecular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of
Health, 5 Research Court, Rockville, MD 20850;†Section on Structural Cell Biology, Laboratory of Cell Biology, National Institute on Deafness and
Other Communication Disorders, National Institutes of Health, 50 Convent Drive, Bethesda, MD 20892; and§Department of Molecular Biology,
Princeton University, Princeton, NJ 08544
Edited by Jeremy Nathans, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 3, 2002 (received for review
July 18, 2002)
Deafness in spontaneously occurring mouse mutants is often
associated with defects in cochlea sensory hair cells, opening an
avenue to systematically identify genes critical for hair cell struc-
ture and function. The classical semidominant mouse mutant
varitint-waddler (Va) exhibits early-onset hearing loss, vestibular
defects, pigmentation abnormalities, and perinatal lethality. A
second allele, VaJ, which arose in a cross segregating for Va, shows
a less severe phenotype. By using a positional cloning strategy, we
identify two additional members of the mucolipin gene family
(Mcoln2 and Mcoln3) in the 350-kb VaJminimal interval and
provide evidence for Mcoln3 as the gene mutated in varitint-
waddler. Mcoln3 encodes a putative six-transmembrane-domain
protein with sequence and motif similarities to the family of
nonselective transient-receptor-potential (TRP) ion channels. In the
Va allele an Ala419Pro substitution occurs in the fifth transmem-
brane domain of Mcoln3, and in VaJ, a second sequence alteration
(Ile362Thr) occurring in cis partially rescues the Va allele. Mcoln3
localizes to cytoplasmic compartments of hair cells and plasma
membrane of stereocilia. Hair cell defects are apparent by embry-
onic day 17.5, assigning Mcoln3 an essential role during early hair
cell maturation. Our data suggest that Mcoln3 is involved in ion
homeostasis and acts cell-autonomously. Hence, we identify a
molecular link between hair cell physiology and melanocyte func-
tion. Last, MCOLN2 and MCOLN3 are candidate genes for heredi-
tary and?or sporadic forms of neurosensory disorders in humans.
ment of the endolymph through melanocytes located in the stria
vascularis; deflection of stereocilia situated on the apical surface
of sensory hair cells in the organ of Corti (OC); opening of
mechanosensitive transduction channels, nonselectively perme-
able for cations, including Ca2?; and transmission of the elec-
trical impulse onto the eighth cranial nerve for further process-
ing in the auditory brainstem and cortex. Mutations affecting
these processes are often associated with circling behavior,
ataxic movements, and pigmentation anomalies. Circling and
waltzing phenotypes are usually caused by mutations primarily
affecting structure and function of hair cells, which, directly or
indirectly, lead to stereocilia disorganization and hair cell de-
generation. Genes critical for hair cells encode a functionally
heterogenous group of proteins that include structural proteins
(myosins, cadherins, and spectrins), extracellular matrix proteins
(tectorins), transmembrane proteins (channels, pumps, ion
the many coat color and spotting mutants, dominant spotting
(KitW), steel (KitlSt), microphthalmia (Mitfmi), and varitint-
defects. The genes underlying W, St, and mi have been shown to
encode the c-Kit receptor tyrosine kinase (2, 3), its ligand Kitl
(4, 5), and the Mitf transcription factor (6), respectively. Muta-
tions in Kit and Mitf impair the survival of melanocytes in the
erception and transmission of acoustic stimuli in the mam-
malian cochlea is a stratified process involving the establish-
stria vascularis, resulting in the loss of the endochochlea poten-
tial, and deafness (7, 8).
To identify genes important for hair cell function, we adopted
a positional cloning approach of mouse mutations with known
defects in the OC. Two mutations, Va and VaJ, arose spontane-
ously at the varitint-waddler locus, causing a distinct allele-
specific set of phenotypes (9–11). Most severely affected are
Va?Va mice, which exhibit deafness, circling behavior, sterility,
mildest phenotype is seen in ??VaJmice, which are viable, show
normal vestibular function, display only limited variegation and
coat color dilution, and have some residual hearing (10, 12, 13).
VaJ?VaJand ??Va mice show intermediate and similar pheno-
types. Hearing tests and anatomical studies associated hearing
loss in VaJmice with a primary defect in the sensory epithelium
and reduced pigmentation in melanocytes located in the stria
vascularis (12). These defects were found to occur indepen-
dently, suggesting that Va acts as a cell-autonomous factor. Thus,
unlike other spotted deaf mouse mutants (Mitfmi, KitW, and
KitlSl), in which neuroepithelial degeneration develops second-
ary to the stria vascularis defects, Va might reveal an interesting
Materials and Methods
Mice and DNA. Mutant and common inbred mouse strains were
obtained from The Jackson Laboratory. B6Fe-a?a-Hoxa13Hd
VaJmice were backcrossed to the C57BL?6J strain for10 gen-
erations and outcrossed to C3HeB?FeJ, and the resulting prog-
eny were intercrossed. RSV?Le-Va?? mice were outcrossed to
C3HeB?FeJ and then intercrossed. Intercrossed offspring from
Va and VaJstrains were used for phalloidin staining and immu-
nocytochemistry. Genomic DNA was obtained from The Jack-
with National Institutes of Health guidelines (Animal Study
Physical Map. The physical map was constructed by screening the
ES-129?SvJ I (Incyte Genomics, Palo Alto, CA) and the
C57BL?6J RPCI-23 (Roswell Park Cancer Institute) mouse
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TRP, transient receptor potential; BAC, bacterial artificial chromosome;
IHCs, inner hair cells; OHCs, outer hair cells; OC, organ of Corti; Pn, postnatal day n; En,
embryonic day n.
database (accession nos. AC068974, AC079941, AY083531, AY083532, and AY083533).
See commentary on page 14613.
University Medical Center, 3800 Reservoir Road NW, Washington, DC 20007-2197.
¶Present address: Department of Molecular Pharmacology, Merck Research Laboratories,
Merck & Co., 770 Sumneytown Pike Road, West Point, PA 19486.
?To whom correspondence should be addressed. E-mail: email@example.com.
November 12, 2002 ?
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no. 23 www.pnas.org?cgi?doi?10.1073?pnas.222425399
bacterial artificial chromosome (BAC) libraries. We screened
the ES-129?SvJ I BAC library by PCR with the flanking
recombinant markers (D3Mit85, D3Mit260, D3Mit259, and
D3Mit292). Probes from the BAC ends of the ES-129?SvJ I BAC
clones were used to screen the RPCI-23 library by hybridization.
BAC clones were sized by restriction endonuclease digestion
with NotI (NEB) followed by pulsed-field gel electrophoresis
(PFGE) on a Chef-DRII apparatus (Bio-Rad). We sequenced
BAC ends with vector-specific primers by using BigDye termi-
(Applied Biosystems). To confirm the chromosomal location of
each identified clone and to establish the orientation of Sp6 and
T7 ends, we developed sequence-tagged sites (STSs), primer
sets, and probes from the end sequences of BAC clones and
cross-screening experiments and Southern blot hybridizations.
Overlaps among individual BAC clones were confirmed by
fingerprinting with HindIII and?or EcoRI single or double
Gene Identification and Mutation Analysis. Draft sequences from
RP23-108E10 and RP23-121J1 BAC clones were generated by
the Department of Molecular Genetics, Albert Einstein College
of Medicine Genome Center, through the TRANSNIH BAC
Sequencing Program (www.nih.gov?science?models?bacse-
quencing) and released into GenBank (accession nos. AC068947
and AC079941). BLAST searches of the EST database with these
genomic sequences identified a cluster of mouse ESTs with high
similarity to the full-length human cDNA clone AK001868. To
obtain the homologous mouse sequence, we sequenced the
longest EST clones AI787597 and AA756265. We designed
primers and performed RT-PCR on C57BL?6J-derived brain
cDNA to establish the mouse Mcoln3 cDNA sequence (Gen-
Bank accession no. AY083531). We deduced its genomic struc-
ture by aligning the mouse and human cDNA against genomic
sequences by using BLASTN searches and DNA-Pustell matrix
alignments (MACVECTOR Version 6.0, Oxford Molecular). To
identify mutations, genomic DNA from wild-type (C57BL?10J
and C57BR?cdJ) and mutant (Va?Va and VaJ?VaJ) strains was
used to amplify by PCR exon-specific products by using primer
pairs complementary to flanking intronic sequences. PCR was
carried out by using the Advantage cDNA polymerase mix
(CLONTECH) according to the manufacturer’s instructions.
PCR products were gel purified following the Qiagen (Chats-
worth, CA) gel extraction kit protocol and sequenced by using
the BigDye chemistry on an ABI 377 automated sequencer.
Wild-type and mutant sequences were compared by using
SEQUENCHER Version 3.0 (Gene Codes, Ann Arbor, MI) software.
Primers to amplify across the Va 1255G 3 C mutation are as
follows: 5?-1138-TCAACTATGCTCGTGTGGC-3? (forward),
Primers to amplify across the VaJ1085T 3 C mutation are as
follows: 5?-970-CACTACAAGAAGGAAGTTTCGG-3? (for-
(reverse). To identify mutations in ??VaJand VaJ?VaJ-derived
cDNA, poly(A)?RNA was isolated from the brain and spleen
according to the Oligotex Direct mRNA kit protocol (Qiagen),
and reverse transcribed with an oligo(dT) primer by using the
SuperScript Preamplification System (Invitrogen). RT-PCR was
carried out and resulting products were directly sequenced.
Phalloidin Staining. Cochlear ducts were dissected in Leibovitz’s
L-15 medium (Invitrogen) from wild-type, VaJ, and Va mice.
Ducts were fixed in 4% paraformaldehyde for 2 h at room
temperature and microdissections were performed to isolate the
OC. Tissues were permeabilized in 0.5% Triton X-100 in PBS for
30 min, washed twice in PBS for 10 min, and then stained with
min. After three 10-min washes in PBS, samples were mounted
by using the ProLong Antifade kit (Molecular Probes) and
examined with a laser scanning confocal microscope (LSM 510,
Immunocytochemistry. To obtain antibodies against Mcoln3
(GenBank accession no. AY083531), rabbits were immunized
(Covance Research Products, Denver, PA) against synthetic
peptide 1, NH2-446-RVSECLFSLINGDDMFS-COOH, and
peptide 2, NH2-529-KDLPNSGKYRLEDDPPGSLL-COOH
(Princeton Biomolecules, Langhorne, PA). Immunocytochem-
istry was performed as described (14). OCs were dissected as
described above. After permeabilization in 0.5% Triton X-100
for 30 min, samples were washed three times in PBS for 10 min
and incubated in 5% normal goat serum (Life Technologies,
Grand Island, NY) and 2% BSA (ICN) in PBS for 2 h to block
nonspecific binding sites. After incubation with primary anti-
bodies at 3–6 ?g?ml in blocking solution for 2 h at room
temperature, samples were washed several times in PBS and
incubated in a 1:200 dilution of the fluorescein-conjugated
anti-rabbit IgG secondary antibody (Amersham Pharmacia Bio-
tech, Uppsala) for 40 min. Samples were mounted by using the
ProLong Antifade kit and examined with a laser scanning
confocal microscope (LSM 510, Zeiss). Preimmune sera and
preincubation of primary antibody with an excess of immuno-
stained for actin with rhodamine-conjugated phalloidin. We
obtained the same results (Fig. 4) with affinity-purified (Co-
vance Research Products) antisera PB221 and PB220 raised
against peptide 1, and antisera HL4559 and HL4560 raised
against peptide 2 (Fig. 4).
GenBank Accession Numbers. Mouse mucolipin-1 (Mcoln1),
AF302009; human mucolipin-1 (MCOLN1), AF249319;
mouse mucolipin-2 (Mcoln2), AY083532; human mucolipin-2
(MCOLN2), AY083533; mouse mucolipin-3 (Mcoln3),
AY083531; human mucolipin-3 (MCOLN3), AF475085; Dro-
sophila CG8743, AAF49118; Caenorhabditis elegans CUP-5,
AF338583; Pkd2, AF014010; and TRP, JU0092. Genomic clones
containing Mcoln2 and Mcoln3, AC068947 (RP23-108E10), and
Perinatal Cochlea Hair Cell Defects in Va and VaJMutants. Previous
ultrastructural studies showed hair cell degeneration in 14-day-
old [postnatal day 14 (P14)] VaJmutants (12). To determine the
onset and extent of the hair cell defects in both alleles, we
examined phalloidin-stained whole mount OC preparations with
scanning laser confocal microscopy. The first signs of abnormal-
ities were detected in VaJ?VaJand as early as embryonic day 17.5
(E17.5), when the growing microvilli of both inner hair cells
(IHCs) and outer hair cells (OHCs) show an irregular arrange-
ment (Fig. 1D). Occasionally, gaps were observed in the row of
IHCs, indicating signs of degeneration (Fig. 1D, arrow). At P5,
stereocilia bundles of IHCs and OHCs seem to have grown to
their normal length but they appear in small clumps at the top
of some hair cells, showing a disorganized pattern (Fig. 1H)
rather than the normal arrangement observed in littermate
controls (Fig. 1E). Stereocilia disorganization in both IHCs and
OHCs continued to progress from P5 to P11 when many of them
appear missing through the length of the OC, and extensive
fusion and clumping are apparent (Fig. 3L, arrowhead). We also
examined HCs at the level of the cuticular plate but no irregu-
larities were detected; occasionally, however, we noticed an
extrusion of hair cell bodies through the reticular lamina and an
shown). In ??Va we observed a similar progressive disorgani-
zation, with stereociliar bundles of both IHCs and OHCs
Di Palma et al.
November 12, 2002 ?
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becoming equally disorganized at all time points (Fig. 1 C, G,
and K). The least severe pathology is seen in ??VaJ, in which the
general organization of the microvilli appears mostly normal at
E17.5 (Fig. 1B). However, disruption of the stereocilia bundle is
apparent by birth and mainly in IHCs, where disorganization of
the bundle remains severe throughout development (Fig. 1 F and
J). Only some minor signs of disorganization are noticeable in
the OHC bundles at P5 (Fig. 1F) but stereocilia disorganization
continues to progress, becoming more pronounced by P11 (Fig.
1J). The progression of hair cell defects from base to apex, along
its developmental gradient, and the perinatal expression of the
phenotype attributes to Va a critical role during early steps of
hair cell differentiation.
The VaJCritical Interval Spans 350 kb and Contains Three Genes.
Previous linkage analyses placed Va to distal chromosome 3
(15), and we recently refined the VaJmap position to a
0.14-centimorgan interval (13). To clone Va, we established a
physical map with BAC clones, spanning an estimated physical
distance of not more than 350 kb, across the VaJrecombinant
interval (Fig. 2). Two overlapping BAC clones (RP23–121J1
and RP23–108E10), positive for the recombinant flanking
markers, were chosen for sample sequencing. Homology
searches with genomic sequences from these clones identified
the previously known lysophosphatidic acid receptor gene,
Edg7 (16), a full-length mouse mRNA AK014467, and several
overlapping mouse ESTs representing a cDNA not previously
described. AK014467 and the mouse transcript shared signif-
icant amino acid similarities (67% and 73%, respectively)
to human mucolipin-1 (MCOLN1, AF249319) and were sub-
sequently designated Mcoln2 (AY083532) and Mcoln3
Mucolipin-3 (Mcoln3) Encodes a Predicted Ion Channel. Mcoln3 has
an ORF of 1,662 bp and encodes a protein of 553 aa with a
predicted molecular mass of ?64 kDa (Fig. 5, which is
published as supporting information on the PNAS web site,
www.pnas.org). The deduced amino acid sequence shares the
highest degree of similarity with Mcoln2 (77%) and Mcoln1
(74%). Secondary structure analyses predict that Mcoln3
Labeling of cochlear hair cells F-actin by FITC-phalloidin at E17.5 (A–D), P5
(E–H), and P11 (I–L). Shown are optical sections through the OC at the level
of the stereocilia. Normal hair cells can be distinguished by E17.5 as a
regular array of microvilli from which ordered rows of stereocilia bundles
develop; in wild-type mice, hair cell stereocilia are V-shaped in OHCs (top
three rows), and straight in IHCs (bottom row) (A, E, and I). In ??VaJ
mutants hair cell bundles appear mostly normal at E17.5 (B); disorganiza-
tion of IHC stereocilia is severe by birth and continues to progress from P5
(F) to P11 (J); only minor signs of disorganization are noticeable in OHC
bundles by P5 (F), but disorganization of the OHC bundle continues to
progress, becoming more pronounced by P11 (J). In ??Va mutants, IHC and
OHC stereociliar bundles are equally disorganized at all time points (C, G,
and K). In VaJ?VaJmutants, stereocilia disorganization is apparent in both
IHCs and OHCs at E17.5 (D), and progresses from P5 (H) to P11 (L). In all
mutant genotypes, by P11 many stereocilia appear missing and extensive
fusion and clumping are apparent (J–L, arrowheads). The spatial organi-
zation of hair cells in the OC in all mutants remains unaltered with three
rows of OHCs and one row of IHCs. (Bars, 10 ?m.)
Development of cochlear hair cells in varitint-waddler mutants.
SP6 and T7 ends of BACs are indicated by S or T, respectively. BAC ends used to derive probes for screening of the RPCI-23 BAC library are indicated by ^. Dashed
lines in red indicate anchored SP6 and T7 BAC ends that have been used in PCR cross-screening and?or Southern blot experiments to confirm overlaps among
BAC clones. Genes that map within the candidate region are shown with an arrow indicating transcription orientation. Mcoln3 exons are indicated. Genomic
clones containing Mcoln2 and Mcoln3, AC068947 (RP23-108E10), and AC079941 (RP23-121J1).
www.pnas.org?cgi?doi?10.1073?pnas.222425399Di Palma et al.
contains six transmembrane domains (S1–S6) with short
cytoplasmic amino and carboxy termini (Fig. 3A). Sequence
motif analyses identify an ion-transport domain (Pfam00520)
and a transient-receptor-potential-like (TRPL) motif
(PS50272) between S3 and S6, as well as a putative pore region
between S5 and S6 (PS50273) (Fig. 3B). Sequence similarities
of Mcoln3 with Drosophila CG8743 (63%) and C. elegans
CUP-5 (55%) homologues are significant, with the highest
degree of conservation being observed across the TRPL motif
(74% and 73%, respectively). Amino acid sequence compar-
ison of Mcoln3 with Pkd2 and TRP reveals only limited but
significant similarity (49% and 32%, respectively) across the
TRPL region (Fig. 3B).
The Varitint-Waddler Phenotype Is Associated with Mutations in
Mcoln3. All 12 coding exons of Mcoln3 were analyzed for
mutations by sequence analyses of PCR products amplified from
the genomic DNA of mutant (Va?Va and VaJ?VaJ) and control
strains (C57BL?10J and C57BL?6J; Fig. 3C). The Va allele
carries a 1255G 3 C transversion in exon 10 leading to an
pore region (amino acids 480–505; in purple) are shown. Mutations in Mcoln3 are indicated by red dots. Blue lines represent regions of Mcoln3 containing
polyclonal antibody recognition sites: PB221 and PB220 antisera were raised against amino acids 446–462, HL4559, and HL4560 against amino acids 529–548.
Pkd2 (amino acids 478–682) and TRP (amino acids 430–665). Putative transmembrane domains (S1–S6) are indicated by thick blue lines. Predicted ion transport
domain and TRPL motif (PS50272) of Mcoln3 are located between amino acids 337–501 and amino acids 317–505, respectively. Conserved amino acids across the
Mcoln3 TRPL region are shaded yellow. Sites of mutations in Mcoln3 are shown in red and in CUP-5 in teal. Amino acid positions are given on the right. (C)
Mutation analysis. Sequence chromatographs showing nucleotide sequence and translation across the sites of mutations in C57BL?10J, Va?Va, and VaJ?VaJ
genomic DNA. Nucleotide changes are shown in red.
Di Palma et al.
November 12, 2002 ?
vol. 99 ?
no. 23 ?
Ala419Pro substitution in the fifth predicted transmembrane
domain of the protein (Fig. 3 A and C). The mutation in VaJis
a 1085T 3 C transition in exon 8 resulting in an Ile362Thr
substitution in the second predicted extracellular loop (Fig. 3 A
change in VaJ, suggesting that VaJarose in cis to Va (Fig. 3C).
These nucleotide changes cosegregated with VaJin the critical
recombinants, and were not present in the parental or other
representative inbred strains. No mutations were found in the
coding regions of Edg7 and Mcoln2.
Mcoln3 Localizes to Vesicular Compartments and Stereocilia in
Cochlea Hair Cells. To gain insights into the cellular function of
Mcoln3, we determined its subcellular location. By using poly-
clonal antibodies directed against either an extracellular or
cytoplasmic epitope of Mcoln3 (Fig. 3A), the distribution of the
protein in the OC was determined in wild-type (Fig. 4A) and
mutant mice (Fig. 4 D and E), and adult rat (Fig. 4 F–J). In the
mouse, Mcoln3 is highly expressed in the cytoplasm of IHCs and
OHCs where labeling appears as a punctuate pattern throughout
the hair cell bodies (Fig. 4A). A similar staining was also
observed in the cytoplasm of IHCs and some OHCs of VaJ?VaJ
and ??VaJmice (Fig. 4 D and E). Immunoreactivity in the adult
OC of the rat was specific and concentrated to cytoplasmic
generated (Fig. 4 F–J). Fluorescence labeling was also detected
in the subcuticular region (Fig. 4I) and the pericuticular necklace
(Fig. 4J). In addition, modest immunoreactivity localizes to the
plasma membrane of the stereocilia (Fig. 4J). Consistent with
circling behavior in Va and VaJmutants, we also detected Mcoln3
expression in vestibular hair cells (data not shown).
A high-resolution genetic and physical mapping approach local-
ized Va to a minimal region of 350 kb, in which three genes were
identified. The only nucleotide changes were found in Mcoln3,
and those were absent from parental controls (C57BR?cdJ,
C57L?J, C58?J, C57BL?6J, C57BL?10J, C57BL?KS, C57BL?
ScSnJ, and STOCK-a?a ma ft?ma ft) and in other strains that are
representative members of different subgroups of inbred mouse
strains (17) such as Castle’s mice (CBA?CaJ, 129?SvJ, DBA?2J,
BALB?cByJ, and AKR?J), Swiss mice (FVB?FnJ and NOD?
MOLF?Ei, CZECHII?Ei, and CAST?Ei) suggesting that the
sequence alterations are causative mutations. Moreover, similar
pathogenic mutations were found in Cup-5 (G401E and G473D)
and MCOLN1 (D362Y and V446L). The Ala419Pro substitution
in Va in the fifth transmembrane domain, located near the
predicted pore region, is likely to cause a gain-of-function or
dominant-negative effect (18), although other mechanisms, such
as the partial loss of functionality, cannot be entirely ruled out.
VaJwas first recognized as a less variegated offspring from a Va
linkage test cross, and subsequent breeding showed that the
deviant was a somatic and germ-line mosaic and allelic to Va
(10). The presence of two missense mutations in Mcoln3 in
VaJargues that the 1085T 3 C transition arose in cis to Va.
Given the consistently milder phenotypes in VaJ, it suggests that
Ile362Thr acts as an intragenic suppressor mutation. However,
definitive proof of the causative nature of the missense muta-
tions awaits further transgenic experiments and molecular func-
Mucolipins constitute a newly recognized family of cation
channel proteins with homologues in mouse (Mcoln1), D. mela-
nogaster (CG8743), and C. elegans (CUP-5). Mutations in human
MCOLN1 cause the neurodegenerative lysosomal disorder mu-
colipidosis type IV (OMIM 252650) (19–22), and loss-of-
function mutations in C. elegans-CUP-5 lead to endocytosis
defects, formation of large lysosomal vacuoles, and increased
apoptosis (23, 24). Here, we describe two additional members,
Mcoln2 and Mcoln3, of the mammalian gene family. The actual
or genetic means. The motif and sequence similarities of Mcoln3
with C. elegans CUP-5 and human MCOLN1 as well as the
punctuate staining of cytoplasmic compartments and the per-
cuticular necklace suggest that Mcoln3 is associated with vesic-
ular structures (25), and thus plays a critical role in vesicles.
Defects in vesicle function often result in skin pigmentation
abnormalities such as seen in the dilute, leaden, chocholate, or
ashen mutants in which mutations in myosin Va (MyoVa),
melanophilin (Mlph), Rab38, and Rab27a affect transport and
trafficking of late-stage melanosomes (26–29). Based on the
motif similarities with ion channels, we hypothesize that Mcoln3
is involved in vesicular and?or intracellular ion homeostasis in
inner ear hair cells and melanocytes.
Shown are cross-sections through the OC showing the labeling of IHCs for
Mcoln3. (A–E) Mouse OC at P11, and optical sections at the level of the cell
body of IHCs. In A a single row of IHCs and three rows of OHCs (1, 2, and 3) are
presented. (A, D, and E) PB221 labeling for Mcoln3. The cytoplasm of IHCs is
intensively labeled in wild type (A), ??VaJ(D), and VaJ?VaJ(E); staining is
(C). Some labeling is also observed in the cytoplasm of OHCs (A, D, and E), and
under the cuticular plate (not shown). (F–J) Rat OC at 4 weeks of age,
and optical sections at the level of cell bodies (F–H) and the cuticular plate (I
(G), and HL4460 (H) showed immunoreactivity in the cytoplasm of IHCs. In the
cytoplasm, labeling appears as a punctuate pattern (*, I) throughout the IHC
bodies, and it is also detected in the subcuticular region (dashed lines, I), the
pericuticular necklace (white arrows, J), and in the IHC bundles (white arrow-
heads, J). Specificity of all four antisera was confirmed by immunoblot after
bacterial expression of a partial Mcoln3 fusion protein (data not shown).
Stereocilia staining was also observed on mouse OC (data not shown). (Bars,
Localization of Mcoln3 in mouse and rat OC by immunofluorescence.
www.pnas.org?cgi?doi?10.1073?pnas.222425399Di Palma et al.
Va homozygotes were reported to be less viable (9), and we
recently showed that VaJhomozygotes on the C57BL?6J back-
ground (after six backcross generations) have only a 34% chance
of survival (13). Possibly, embryonic melanocytes fail to differ-
entiate, migrate, or survive, and subsequently, downstream
apoptotic events, similar to those observed in the null allele of
CUP-5 (24), may cause the embryonic lethal phenotype. This
observation and our generation of a C57BL?6J congenic strain
(B6.VaJ) present an additional avenue to further characterize the
cellular function of Mcoln3 and its dependence on the genetic
Mucolipins show sequence and motif similarities to the TRP
ion channel family (30–32), including the polycystin-2 (PKD2)
protein family of nonselective intracellular ion channels (33–35).
TRP channels are characterized by a six-transmembrane-domain
topology and their permeability to cations, including Ca2?(31),
a characteristic that is shared with the mechanosensitive trans-
duction channel in vertebrate hair cells. Proteins of the TRP
family are involved in the transduction of sensory stimuli in
vision (36), olfaction (37), thermoreception (38), osmoregula-
tion (39), and mechanosensation (40). Two members of the TRP
gene family, NompC and TRPV4 (former Osm-9), were shown to
be involved in mechanotransduction in D. melanogaster and
C. elegans, respectively (37, 40), and more recently the murine
homolog of TRPV4 was localized to inner ear hair cells (41). Our
localization of Mcoln3 to the stereocilia, however, leads to the
interesting hypothesis that this predicted channel might also be
involved in sensory transduction.
Several lines of evidence argue for a cell-autonomous function
of Mcoln3: (i) independent defects in the OC and stria vascularis
as shown (12), (ii) erratic circling behavior indicative of vestib-
ular hair cell defects, and (iii) localization of Mcoln3 to cochlea
and vestibular hair cells. Thus, Va encodes an interesting mo-
lecular link between inner ear hair cells and melanocytes.
Finally, human MCOLN2 and MCOLN3 are candidate genes for
inherited and?or sporadic neurosensory disorders.
We thank Bill Pavan, Mark Fogg, Feng Qian, Emma Morton-Bours,
Keith Vokey, and Susan Cole for discussions, Doris Wu, Dennis Drayna,
Bob Wenthold, and Heinz Arnheiter for valuable comments on the
manuscript, Alfredo Calderon for technical assistance, and Melissa Irby
and Jimmy Fiallos for animal colony management. This work was
supported by the intramural program of the National Institute on
Deafness and Other Communication Disorders.
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