Induction
of
calcium
influx
through
TRPC5
channels
by
cross
-
linking
of
GM1
ganglioside
associated
with
alpha5beta1
integrin
initiates
neurite
outgrowth
.
Previous
studies
demonstrated
that
cross
-
linking
of
GM1
ganglioside
with
multivalent
ligands
,
such
as
B
subunit
of
cholera
toxin
(
CtxB
)
,
induced
Ca2+
influx
through
an
unidentified
,
voltage
-
independent
channel
in
several
cell
types
.
Application
of
CtxB
to
undifferentiated
NG108-15
cells
resulted
in
outgrowth
of
axon
-
like
neurites
in
a
Ca2+
influx
-
dependent
manner
.
In
this
study
,
we
demonstrate
that
CtxB
-
induced
Ca2+
influx
is
mediated
by
TRPC5
channels
,
naturally
expressed
in
these
cells
and
primary
neurons
.
Both
Ca2+
influx
and
neurite
induction
were
blocked
by
TRPC5
small
interfering
RNA
(
siRNA
)
.
Pretreatment
of
NG108-15
cells
with
neuraminidase
increased
cell
-
surface
GM1
and
greatly
enhanced
the
signal
.
GM1
was
not
directly
associated
with
TRPC5
but
rather
with
alpha5beta1
integrin
,
which
opened
the
channel
through
a
signaling
sequence
after
cross
-
linking
of
the
GM1
/
integrin
complex
.
This
cascade
included
autophosphorylation
of
focal
adhesion
kinase
and
subsequent
activation
of
phospholipase
Cgamma
(
PLCgamma
)
and
phosphoinositide-3
kinase
[
PI(3)K
]
.
Pharmacological
blockers
that
inhibited
tyrosine
kinase
,
PLC
,
and
PI(3)K
suppressed
both
CtxB
-
induced
Ca2+
influx
and
neurite
outgrowth
.
These
were
also
suppressed
by
SK&amp;F96365
,
a
nonspecific
transient
receptor
potential
channel
blocker
.
Confocal
immunocytochemistry
revealed
that
GM1
cross
-
linking
induced
colocalization
of
GM1
with
these
signaling
elements
in
sprouting
regions
of
plasma
membrane
.
In
primary
cerebellar
granular
neurons
(
CGNs
)
,
TRPC5
was
detected
at
2
d
in
vitro
(
2
DIV
)
,
a
stage
corresponding
to
CtxB
-
stimulated
Ca2+
influx
.
Neurite
outgrowth
in
CGNs
,
determined
at
3
DIV
,
was
accelerated
by
CtxB
and
suppressed
by
TRPC5
siRNA
and
the
above
blockers
.
The
crucial
role
of
GM1
was
indicated
with
CGNs
from
ganglio
-
series
null
mice
,
in
which
growth
of
axons
was
significantly
retarded
.