C-termini of P/Q-type Ca21channel a1A subunits
translocate to nuclei and promote
Holly B. Kordasiewicz1,2, Randall M. Thompson1,2, H. Brent Clark2,3
and Christopher M. Gomez1,2,*
1Department of Neuroscience,2Department of Neurology and3Section of Neuropathology, University of Minnesota,
420 Delaware Street SE, Minneapolis, MN 55455, USA
Received November 12, 2005; Revised and Accepted March 24, 2006
P/Q-type voltage-gated calcium channels are regulated, in part, through the cytoplasmic C-terminus of their
a1A subunit. Genetic absence or alteration of the C-terminus leads to abnormal channel function and neuro-
logical disease. Here, we show that the terminal 60–75 kDa of the endogenous a1A C-terminus is cleaved
from the full-length protein and is present in cell nuclei. Antiserum to the C-terminus (CT-2) labels both wild-
type mouse and human Purkinje cell nuclei, but not leaner mouse cerebellum. Human embryonic kidney cells
stably expressing b3 and a2d subunits and transiently transfected with full-length human a1A contain a
75 kDa CT-2 reactive peptide in their nuclear fraction. Primary granule cells transfected with C-terminally
Green fluorescent protein (GFP)-tagged a1A exhibit GFP nuclear labeling. Nuclear translocation depends
partly on the presence of three nuclear localization signals within the C-terminus. The C-terminal fragment
bears a polyglutamine tract which, when expanded (Q33) as in spinocerebellar ataxia type 6 (SCA6), is
toxic to cells. Moreover, polyglutamine-mediated toxicity is dependent on nuclear localization. Finally, in
the absence of flanking sequence, the Q33 expansion alone does not kill cells. These results suggest a
novel processing of the P/Q-type calcium channel and a potential mechanism for the pathogenesis of SCA6.
Spinocerebellar ataxia type 6 (SCA6) is a disorder of progress-
ive cerebellar dysfunction and is one of at least three domi-
nantly inherited neurological disorders caused by mutations in
the CACNA1A gene (1,2). The CACNA1A gene encodes the
a1A subunit, the transmembrane pore-forming subunit of the
P/Q-type or CaV2.1 voltage-gated calcium channel (VGCC)
(2). Whereas the other CACNA1A disorders are associated
with simple missense, truncating or splicing mutations,
SCA6, such as Huntington’s disease (HD) (3) and other
forms of SCA (4–6), is caused by abnormal expansion of a
trinucleotide CAG repeat encoding an elongated tract of gluta-
mine residues. In SCA6, the expansion is found in exon 47 of
the CACNA1A gene, which normally contains a polymorphic
CAG repeat tract (CAG)4–18encoding 4–18 glutamines in the
C-terminus of the a1A subunit, but is expanded to the patho-
logical range of (CAG)19–33, encoding 19–33 glutamines (7,8).
VGCC are multimeric complexes composed of at least three
protein subunits: a pore-forming subunit (a1) and two auxili-
ary subunits (b and a2d). Distinct genes encode more than
10 different a1 subunits (a1A–I,S), which confer different
channel properties and are expressed in different cell types
mutations in the various VGCC genes (1,2). P/Q channels
are involved in a diverse array of cell functions including neu-
rotransmitter release, regulation of gene expression, release of
calcium from internal stores and dendritic calcium transients
(9–12). P/Q channels are highly expressed in cerebellar
neurons and localize primarily to nerve terminals, dendrites
and Purkinje cell soma (11,13).
The full-length a1A subunit contains four homologous
transmembrane repeat domains (I–IV) flanked by three intra-
cellular loops (LI–II, LII–III, LIII–IV) and cytoplasmic N- and
C-termini (9). Complementary DNA sequencing and protein
immunoblot studies with domain-specific antisera have
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*To whom correspondence should be addressed at: Department of Neurology, AMB S237, MC2030, The University of Chicago, 5841 S. Maryland,
Chicago, IL 60637, USA. Tel: þ1 7737026390; Fax: þ1 7737025670; Email: email@example.com
Human Molecular Genetics, 2006, Vol. 15, No. 10
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demonstrated the presence of a1A polypeptides of a range of
sizes and domain compositions arising from alternative spli-
cing and possible proteolytic processing (14–16). Several
studies have suggested the presence of N-terminal fragments
of the a1A subunit that lack portions of the distal C-terminus
ranging from 40 kDa to the entire last two transmembrane
repeat domains (17–19). Conversely, a 75 kDa C-terminal
fragment has been detected in protein extracts of full-length
a1A-expressing human embryonic kidney (HEK) cells
(17,20). These observations suggest that, at least in heter-
ologous systems, the a1A C-terminus is cleaved and may
form a stable a1A polypeptide.
The a1A C-terminus participates in a number of protein–
protein interactions and plays a prominent role in modulating
channel activity (21). Thus, its alteration by proteolytic clea-
vage or genetic mutation would have significant functional
consequences. Mice homozygous for the leaner (Tgla)
mutation, which express a1A subunits lacking the distal
C-terminus, are severely ataxic, exhibit Purkinje cell degener-
ation and die if unattended at ?21 days (22).
In this study, we explored, in the cerebellum, the distri-
bution of putative endogenous C-terminal polypeptides using
an antibody we raised against an a1A C-terminal epitope.
We found that the C-terminal a1A antiserum labels Purkinje
cell nuclei and that a 60–75 kDa proteolytic fragment of the
endogenous a1A C-terminus is present in cell nuclei. Using
plasmid vectors expressing recombinant human a1A protein,
we identified three nuclear localization signals (NLSs).
Finally, because the cleaved C-terminal fragment bears a poly-
glutamine tract, which is expanded in SCA6, we tested
whether expansion of the polyglutamine tract correlated with
cell death. Cells expressing C-terminal proteins bearing
polyglutamine tracts of 33 glutamines had more than twice
the rate of cell death than those expressing unexpanded
normal tracts. Furthermore, this polyglutamine-mediated cell
death appears to be dependent on nuclear localization of the
C-terminal fragment. These results suggest that the a1A
C-terminal cleavage product may play a role in nuclear signal-
ing and in the pathogenesis of SCA6.
The C-terminus of the a1A subunit is present
in cell nuclei
To determine the subcellular distribution of the a1A subunit in
human cerebellar cortex, we used affinity-purified, anti-
peptide antibodies, specific for either the N-terminus (NT-1
antibody) or the C-terminus (CT-2 antibody) of the a1A
subunit. Surprisingly, in paraffin-embedded human cerebellar
tissue, the C-terminal antibody intensely stained Purkinje
cell nuclei (Fig. 1A, inset). CT-2 nuclear staining was not
observed in granule cells or any other cerebellar cell type.
NT-1 and CT-2 antibody also labeled the Purkinje cell mem-
brane, projections and cytoplasm as previously reported for
other a1A antibodies (13,23,24) (Fig. 1A and B). NT-1 anti-
bodies did not label cell nuclei (Fig. 1B), although they did
intensely stain Purkinje cell membranes. A punctate staining
pattern was observed at the cell membrane with both NT-1
and CT-2 antibodies (Fig. 1A and B, inset). Immunostaining
by both NT-1 and CT-2 was specifically blocked by
preincubation with their respective peptide immunogens
(Fig. 1C and D), but not with other peptides (data not
shown). An identical pattern of immunolabeling was seen in
adult mouse cerebellar sections (Fig. 1E and F).
As a further test of specificity, we compared immunostain-
ing of cerebellar sections from homozygous leaner (Tgla)
pups, which express mutant a1A subunits lacking a
C-terminus and thus the CT-2 epitope, with age-matched post-
natal day 18 (P18) control pups. CT-2 antibody failed to label
Purkinje cells from leaner cerebellum, either in the cytoplasm
or in the nucleus (Fig. 1G). NT-1 antibody stained leaner cer-
ebellum in the same pattern as control cerebellum, although
slightly less intensely (Fig. 1H and J). In control P18 cerebel-
lum, CT-2 antibody labeled nuclei more intensely than wild-
type (WT) adult nuclei, and both NT-1 and CT-2 antibody
labeled WT P18 Purkinje cell soma (Fig. 1I and J, inset).
Cell membranes and projections were stained relatively less
Figure 1. The a1A C-terminus is present in Purkinje cell nuclei in mouse and
human cerebellum. Cerebellar sections stained using immunoperoxidase
(brown) with either anti-CT-2 (A, C, E, G and I) or anti-NT-1 (B, D, F, H
and J).Bluenuclearcounterstainis hematoxylin.Paraffin-embeddedcerebellar
sections were from adult human (A and B); adult human blocked by pre-
incubation with the respective peptide (C and D), WT adult mouse (E and
F), homozygous leaner mouse (G and H) and P18 WT mouse (I and J).
Insets are enlargements of Purkinje cell staining. White arrows indicate Pur-
kinje cell nuclei. Black arrows indicate Purkinje cell projections and cell
soma. Scale bars: 75 mm.
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