L-Type Calcium Channels in Insulin-Secreting Cells: Biochemical Characterization and Phosphorylation in RINm5F Cells
Hasan Safayhi,
Hannelore Haase,
Ursel Kramer,
Andrea Bihlmayer,
Monika Roenfeldt,
Hermann P.T. Ammon,
Monika Froschmayr,
Tara N. Cassidy,
Ingo Morano,
Michael K. Ahlijanian and
Jörg Striessnig
Pharmazeutisches Institut, Lehrstuhl Pharmakologie (H.S., U.K.,
A.B., M.R., H.P.T.A.) Universität Tübingen D-72076
Tübingen, Germany
Max Delbrück Centrum für
Molekulare Medizin (H.H., I.M.) Cardiology D-13125 Berlin,
Germany
Pfizer (M.K.A.) Central Research Division
Groton, Connecticut 06340
Institut für Biochemische
Pharmakologie (J.S., M.F., T.N.C.) Universität Innsbruck
A-6020 Innsbruck, Austria
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ABSTRACT
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Opening of dihydropyridine-sensitive
voltage-dependent L-type Ca2+-channels (LTCCs)
represents the final common pathway for insulin secretion in pancreatic
ß-cells and related cell lines. In insulin-secreting cells their
exact subunit composition is unknown. We therefore investigated the
subunit structure of
(+)-[3H]isradipine-labeled LTCCs in
insulin-secreting RINm5F cells. Using subunit-specific antibodies we
demonstrate that
1C subunits (199 kDa, short form) contribute only a
minor portion of the total
1 immunoreactivity in membranes and
partially purified Ca2+-channel preparations.
However,
1C forms a major constituent of
(+)-[3H]isradipine-labeled LTCCs as 54% of
solubilized (+)-[3H]isradipine-binding
activity was specifically immunoprecipitated by
1C antibodies.
Phosphorylation of immunopurified
1C with cAMP-dependent protein
kinase revealed the existence of an additional 240-kDa species (long
form), that remained undetected in Western blots. Fifty seven percent
of labeled LTCCs were immunoprecipitated by an anti-ß-antibody
directed against all known ß-subunits. Isoform-specific antibodies
revealed that these mainly corresponded to ß1b- and ß3-subunits. We
found ß2- and ß4-subunits to be major constituents of cardiac and
brain L-type channels, respectively, but not part of L-type channels in
RINm5F cells. We conclude that
1C is a major constituent of
dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as
a substrate for cAMP-dependent protein kinase. ß1b- and ß3-Subunits
were also found to associate with L-type channels in these cells. These
isoforms may therefore represent biochemical targets for the modulation
of LTCC activity in RINm5F cells.
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INTRODUCTION
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Voltage-dependent Ca2+ channels mediate
depolarization-induced influx of Ca2+ into a variety of
electrically excitable cells, including pancreatic ß-cells. Opening
of these channels represents the final common pathway for insulin
secretion triggered by the major insulin secretagogues glucose, amino
acids, and sulfonylureas (for review see Ref.1). Voltage-dependent
Ca2+ channels therefore represent an important
physiological target for the initiation and modulation of insulin
release (1, 2). Like in neurons and cardiac muscle, major biochemical
pathways of channel modulation include the activation of pertussis
toxin-sensitive G proteins (3, 4, 5, 6) or phosphorylation/dephosphorylation
processes (1, 7, 8).
Based on different biophysical and pharmacological properties, at least
six different channel types can be distinguished. Insulin secretion
from pancreatic ß-cells is tightly coupled to Ca2+ influx
through so-called L-type channels (LTCCs) (1). This channel type is
activated by strong depolarizations and selectively blocked by low
concentrations of Ca2+ antagonist drugs, e.g.
the dihydropyridines (DHPs) nifedipine or isradipine (9, 10). DHPs
reversibly bind to the LTCC complex in various insulin-secreting cell
lines with high affinity (11, 12) and are potent blockers of insulin
secretion (1, 12, 13).
LTCCs in brain and muscle tissues exist as hetero-oligomeric complexes
of at least an
1-, an
2-
-, and a ß-subunit (9, 14). The
1-subunits form the ion-conducting pore and determine the channels
sensitivity for DHPs. From the six cloned
1-subunit genes (termed
1A through
1E and
1S) only
1C,
1D and
1S participate
in the formation of DHP-sensitive Ca2+ channels (for review
and nomenclature see Ref.15). From the four known ß-subunit
isoforms, only ß1a- (in skeletal muscle) and ß2-subunits (in heart)
have so far been found to be associated with LTCCs (16, 17).
ß-Subunits are critical for
1 function and determine the
properties of the channel in a ß-isoform-specific manner
(18, 19, 20).
Although
1C- and
1D-transcripts (21, 22, 23, 24), as well as transcripts
from mammalian ß-subunits [mainly, ß1b, ß2, and ß3 (24, 25, 26, 27)],
were identified in insulin-secreting cells, no biochemical analysis of
the LTCC complex has yet been reported. Knowledge of the subunit
composition of LTCCs in insulin-secreting cells is an important
prerequisite for the generation of cell lines heterologously expressing
channels with the functional and pharmacological properties of LTCCs in
pancreatic ß-cells. Such cell lines would be of great value to
develop drugs that selectively modulate (neuro)endocrine LTCCs. For
example, ß-cell LTCC activators (28) could be very useful in
stimulating insulin secretion in type II diabetes.
Here we present a biochemical analysis of the subunit composition of
LTCCs in a (neuro)endocrine, insulin-secreting cell line, RINm5F cells.
With the help of subunit-specific antibodies, we demonstrate that
1C-subunits, together with ß- and
2-
-subunits, represent a
major constituent of DHP-labeled LTCCs channels in these cells. From
the four ß-subunit isoforms only ß1b and ß3 were found as part of
the LTCC complexes.
We also demonstrate the existence of
1C in two lengths. Its long
(full- length) form was identified as a substrate for in
vitro phosphorylation by cAMP-dependent protein kinase (cA-PK). As
in neurons and muscle cells, posttranslational processing of
1C may
therefore play an important physiological role for LTCC modulation in
insulin-secreting cells. Our results provide the basis for further
studies assessing the role of these subunits for channel function and
stimulus-secretion coupling in pancreatic ß-cells. ß1b- and
ß3-subunits must be considered the major candidates for coexpression
with
1-subunits to construct stable cell lines expressing
neuroendocrine LTCCs.
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RESULTS
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Different Classes of
1-Subunit Polypeptides Copurify with
(+)-[3H]Isradipine-Labeled LTCC Complexes
To identify polypeptides forming LTCCs in RINm5F cells, we
prepared sequence-directed antibodies against Ca2+ channel
1-,
2-
-, and ß-subunits.
1-Antibodies were directed
against peptides corresponding to a sequence conserved in all known
1-subunits (anti-
1) as well as to a sequence unique for
1C
(anti-
1C). We used these antibodies to identify
1
immunoreactivity copurifying with LTCCs on wheat germ agglutinin
(WGA)-Sepharose (29, 30). Membrane-bound channels were prelabeled with
(+)-[3H]isradipine, an L-type selective Ca2+
channel blocker. In our preparations (+)-[3H]isradipine
labeled 513 ± 33 fmol of receptor sites per mg of membrane
protein (n = 3) with subnanomolar Kd and a
pharmacological profile typical for LTCC (11). Accordingly, the
Ca2+ antagonist (+)-tetrandrine (31) stimulated binding to
131 ± 7% (n = 4) of control values. For channel
purification, (+)-[3H]isradipine- prelabeled membranes
were solubilized with 1% (wt/vol) digitonin. Solubilized binding
activity was adsorbed to WGA-Sepharose and, after extensive washing,
biospecifically eluted with
N-acetyl-D-glucosamine. Specific binding
activity was absent in the column flow-through but increased 7-to
15-fold (range, n = 6) with respect to the digitonin extract in
the column eluate. No specific binding activity was retained on the
resin (not shown), suggesting that complete elution was achieved.
Figure 1
shows that the non-subtype-selective antibody
anti-
1 recognized two diffuse bands in membranes as well as in the
digitonin extract. The bands migrated slightly above and below the
prestained myosin marker, with apparent molecular masses of 204 ±
3 and 174 ± 3 kDa (n = 6), respectively. This staining
pattern was highly reproducible among different membrane preparations
(n = 3). Staining was completely eliminated after the
affinity-purified antibody was blocked with the antigenic peptide (1
µM, Fig. 1A
). The two bands were absent in the column
flow-through but enriched in the eluate with respect to the digitonin
extract (see legend to Fig. 1
).
Molecular cloning studies revealed that
1D and
1C are the major
classes of
1-subunits expressed in insulin-secreting cells (21, 22, 23, 24).
To determine the contribution of
1C-immunoreactivity to the total
anti-
1 staining, the same protein samples were immunostained in
parallel with anti-
1C. As illustrated in Fig. 1B
,
1C-immunoreactivity was only detectable in preparations enriched in
(+)-[3H]isradipine binding activity by WGA-affinity
chromatography (n = 3). Anti-
1C recognized a diffuse band that
always comigrated with the prestained myosin marker as a 199 ± 4
kDa band (n = 3) (Fig. 1B
), where only a fraction of total
1-staining was found. Control experiments performed with recombinant
1-subunits expressed in Saccharomyces cerevisiae (see Ref
32. and legend to Fig. 5
) revealed that under our experimental
conditions, affinity-purified anti-
1 and anti-
1C preparations
(n = 2) recognized
1C with similar intensity. Therefore,
1C
accounts for only a portion of the overall
1-immunoreactivity in
RINm5F cells. This finding is not unexpected because
1D- or
1-subunits associated with non-L-type channels are also expressed in
these cells (8, 21, 33).
In skeletal muscle, heart, and brain the purification of LTCC
1-subunits on WGA-Sepharose is attributed to their association with
a heavily glycosylated
2-
-subunit in the channel complex (34).
Accordingly, an
2-
-subunit with an apparent molecular mass of
165 ± 3 kDa (n = 3) also coeluted from our WGA-Sepharose
column (Fig. 2
). This suggests that
2-
-subunits
are also part of the LTCC complex in RINm5F cells.

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Figure 2. 2-Subunit Immunostaining in RINm5F Cell
Membranes
(+)-[3H]isradipine-binding activity from rabbit skeletal
muscle and RINm5F cells was partially purified by WGA-Sepharose
affinity chromatography as described in Materials and
Methods. The following protein samples were separated on a 8%
polyacrylamide SDS-gel and processed for immunostaining with
affinity-purified anti- 2 antibodies: WGA-Sepharose eluate/skeletal
muscle (EL/S; 0.006 mg/lane); RINm5F membranes (MEM, 0.011 mg/lane);
WGA-Sepharose eluate/RINm5F (EL, 0.005 mg/lane). Nonspecific staining
(+) was determined after preincubation of antibodies with 1
µM antigenic peptide. The migration of molecular mass
markers (200, 94, 68, and 45 kDa) is indicated on the
right. The migration of specifically stained bands
enriched in the WGA-Sepharose eluates is indicated by
arrows.
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1C Is a Major Constituent of the
(+)-[3H]Isradipine-Labeled LTCC Complexes in
RINm5F Cells
To determine which
1-subunit is associated with the
(+)-[3H]isradipine-labeled LTCC complex, we performed
immunoprecipitation experiments with anti-
1C-antibodies. In contrast
to anti-
1, which only recognized denatured
1-subunits (35),
anti-
1C immmunoprecipitates the digitonin-solubilized channel
complex in a concentration-dependent manner (Fig. 3
, A
and C). Saturating concentrations of anti-
1C bound 54 ± 8%
(n = 4) of solubilized channel complexes in RINm5F cells. In
control experiments, anti-
1C immunoprecipitated similar portions of
(+)-[3H]isradipine binding activity from guinea pig or
rabbit heart (79 ± 10; n = 5) and guinea pig brain (69
± 8, n = 3). This suggests that, as in heart and brain,
1C
represents a major constituent of DHP-labeled channels in RINm5F
cells.

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Figure 3. Immunoprecipitation of
(+)-[3H]Isradipine-Binding Activity Solubilized from
RINm5F, Heart, and Brain Membranes
A and B, Immunoprecipitation by anti- 1 (A) and anti-ß (B)
antibodies was carried out as described in Materials and
Methods. Immunoprecipitation is expressed as percent of the
total amount of specific precipitable
(+)-[3H]isradipine-binding activity added to each tube.
Specific precipitable binding activity was determined by polyethylene
glycol-precipitation/filtration as described in Materials and
Methods. Data are given as means ± SE (n
3). Panel C shows typical experiments illustrating the concentration
dependence of immunoprecipitation by anti- 1C and anti-ß. The
curves were obtained by fitting the experimental data to a monophasic
hyperbolic function, yielding a maximal immunoprecipitation of 55 and
67% of total specific binding for anti- 1C and anti-ß,
respectively. An affinity-purified antibody against p-glycoprotein
(anti-pgp-389, 64) was used as a control antibody. This antibody
efficiently immunoprecipitates DHP-photolabeled p-glycoprotein from
multidrug-resistant cells (64) but does not recognize Ca2+
channel subunits. The top x-axis indicates the
concentration of the anti-ß antibody. D and E, Immunoprecipitation of
(+)-[3H]isradipine binding activity by isoform-specific
ß-subunit antibodies: labeled channels were immunoprecipitated by
saturating concentrations of anti-ß2 (0.002 mg/ml), anti-ß3 (0.11
mg/ml), anti-ß1b (0.083 mg/ml), and anti-ß4 (0.093 mg/ml). Data are
given as means ± SE (panel D, n 3) or ±
range (panel E, n = 2). Immunoprecipitation by equivalent
concentrations of control rabbit IgG was subtracted to yield specific
immunoprecipitation. To calculate the percent of ß-subunits
associated with a particular isoform, data were expressed as percent of
the radioactivity specifically immunoprecipitated by anti-ß in the
same tissue.
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ß3- and ß1b-Subunits Are Associated with
(+)-[3H]Isradipine- Labeled LTCCs in RINm5F
Cells
We have previously shown that ß-subunits stabilize
1C in a
high affinity state for DHP Ca2+ antagonists (36). Our
non-isoform-selective anti-ß-subunit antibody (anti-ß) efficiently
precipitated solubilized LTCC activity in RINm5F (57 ± 6%,
n = 4), guinea pig, or rabbit heart (66 ± 12%, range,
n = 3) and guinea pig brain (66 ± 13%, n = 4) membranes in a
concentration-dependent manner (Fig. 3
, B and C). This suggests that
ß-subunits form part of the DHP-labeled channel complex in RINm5F
cells.
To determine which ß-isoform is coupled to
1C in RINm5F cells, we
used isoform-specific antibodies raised against the C-terminal portions
of all known ß-subunit isoforms (ß1b, ß2, ß3, ß4).
ß2-Subunits, together with
1C, are the major component of
DHP-labeled LTCCs in mammalian heart (16). A ß2 cDNA has recently
also been cloned from RINm5F cells. In control experiments, saturating
concentrations of affinity-purified anti-ß2 bound 45 ± 8%
(n = 5) of ß-subunit-associated (+)-[3H]isradipine
binding activity solubilized from guinea pig or rabbit heart membranes
(n = 3, Fig. 3D
). Under the same experimental conditions, no
binding activity above background was recognized by anti-ß2
antibodies in RINm5F cells (Fig. 3D
). In brain also, only a small
portion of channels was associated with ß2. In contrast, anti-ß3
antibodies recognized no labeled channels solubilized from heart but
accounted for 21 ± 6% (n = 5) of channel-associated
ß-immunoreactivity in RINm5F membranes (Fig. 3D
) and a similar
portion (20 ± 3%, n = 8) in brain.
Thus it appeared that the ß-subunit composition of LTCCs in RINm5F
cells resembled brain LTCCs. ß1b and ß4 are also preferentially
expressed in neuronal cells (37, 38). As shown in Fig. 3E
, these two
isoforms together comprise more than 50% of the LTCC ß-subunits in
extracts of whole mammalian brain. In RINm5F cells, affinity-purified
anti-ß1b-antibodies also efficiently immunoprecipitated DHP-binding
activity whereas anti-ß4 was ineffective (Fig. 3E
). Taken together,
the majority of ß-subunits associated with LTCCs in RINm5F cells
corresponded to the ß1b- or ß3-isoform.
The specificity of our antibodies was verified in control experiments
using (+)-[3H]isradipine-labeled skeletal muscle LTCCs,
which only contain the ß1a-isoform. Only anti-ß, but none of our
isoform-specific antibodies (n = 2), specifically
immunoprecipitated the skeletal muscle channel complex. Further
evidence for the specific interaction of anti-ß3 and anti-ß1b
antibodies was obtained in immunoblots. The ß3 polypeptide (apparent
molecular mass, 62 ± 2 kDa, n = 4) was detected in RINm5F,
guinea pig brain, and guinea pig cerebellum membranes, but not in
rabbit skeletal muscle membranes (Fig. 4A
). In some
experiments a very faint band was detected in guinea pig heart
membranes (not shown). Anti-ß1b was less reactive in immunoblots.
However, specific staining of the expected ß1b-polypeptide (85
± 1 kDa; n = 3) could be demonstrated in guinea pig cerebral
cortex after enrichment of ß-subunits by affinity chromatography on a
glutathione-S-transferase (GST) fusion protein with the
1
interaction domain of the
1C subunit (AIDA) (Fig. 4B
). GST-AIDA
binds all ß-subunit isoforms with high affinity and can be used for
ß-subunit purification (Ref. 17; see Materials and
Methods). Although this procedure allowed the specific enrichment
of ß3-subunits (not shown) from RINm5F cells and of ß1b-subunits
from brain (Fig. 4B
), no specific ß1b staining was found in RINm5F
membranes. Therefore, ß1b seems to be expressed at lower densities in
RINm5F cells than in brain.

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Figure 4. ß3- and ß1b-Subunit Immunostaining
A, The following protein samples were separated on a 10%
polyacrylamide SDS-gel and processed for immunostaining with
affinity-purified anti-ß3 antibodies: RINm5F membranes (RIN, 0.07
mg/lane); guinea pig cerebellum membranes (CER, 0.06 mg/lane); guinea
pig brain membranes (BR, 0,05 mg/lane); rabbit skeletal muscle
membranes (SKM, 0.006 mg/lane). The migration of prestained molecular
mass markers (106, 80, 49.5, 32.5, and 27.5 kDa) is indicated on the
right. The migration of the specifically stained
ß3-band is indicated by an arrow. In skeletal muscle
membranes, two bands (indicated by asterisks) were
nonspecifically stained by the second antibody. B, ß-Subunits were
enriched from brain (BR) or RINm5F (RIN) membranes by affinity
chromatography on GST-AIDA as described in Materials and
Methods. Purified fractions extracted from about 1 mg of
starting membrane protein) were separated on a 10% SDS gel and
immunostained with affinity-purified anti-ß1b. The migration of
prestained molecular mass markers and of the ß1b-subunit (85 kDa;
arrow) is indicated. The asterisk
indicates the migration of GST-AIDA, which was nonspecifically stained
as was a faint 70-kDa band. Both were also observed in skeletal muscle
ß-subunit preparations. ß1b staining in RINm5F membranes was absent
in three independent experiments in which ß1b staining in brain was
always observed.
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Phosphorylation of a Long Form of
1C by cA-PK in RINm5F
Membranes
In heart muscle (39, 40, 41, 42) as well as in insulin-secreting
cells (1, 43), LTCC activity is increased by cA-PK. To test whether
1C serves as a substrate for this kinase in vitro, we
immunopurified
1C-subunits from Triton X100 extracts of RINm5F cells
to determine whether its full length form is present and could serve as
a substrate for cA-PK in vitro. As shown in Fig. 5
, a single phosphorylated band was isolated by
sequential immunoprecipitation with anti-
1 and anti-
1C
antibodies. Immunoprecipitation was specific because the band was
absent when antibodies lacking affinity for
1 (e.g.
anti-
2, Fig. 5
) were employed in the first immunoprecipitation step.
Anti-
2-antibodies are unable to immunoprecipitate the complex
because the
2-
-subunit dissociates from
1 in Triton
X-100-containing buffers (44). As anti-
1C was used in the second
immunoprecipitation step, the 240-kDa band must represent a
phosphorylated form of
1C. In three independent experiments, the
electrophoretic mobility of the phosphorylated band was always lower
than prestained myosin and the band recognized by anti-
1C in
immunoblots (Fig. 1B
). To obtain further proof that the phosphorylated
band corresponds to a long form of
1C, we compared its
electrophoretic mobility with a phosphorlyated
1-chimera (calculated
molecular mass = 240 kDa) expressed in Saccharomyces
cerevisiae that contains the anti-
1- and anti-
1C-epitopes
(chimera TM-D in Ref 32, see also legend to Fig. 5
). We have recently
shown (32) that proteolytic processing is absent in Saccharomyces
cerevisiae. The phosphorylated band in RINm5F cells strictly
comigrated with the phosphorylated
1-chimera that was subjected to
the same immunopurification and phosphorylation procedure (Fig. 5
).
This implies that in RINm5F cells proteolytic removal of the C-terminal
portion containing the consensus sites for phosphorylation by cA-PK
(45) takes place in most of the
1C-subunits, giving rise to the
199-kDa form detected in immunoblots.
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DISCUSSION
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Our studies provide the first biochemical analysis of an
LTCC in a (neuro)endocrine cell type on the protein level. Because
biochemical studies in islets of Langerhans would be technically
difficult to perform due to low harvest number and cell type
heterogeneity, we selected RINm5F cells for our studies. This cell line
is widely used to study the role of LTTCs for insulin secretion (see
references in Ref.12). Although RINm5F cells have lost some typical
features of mouse or human pancreatic ß-cells (e.g. the
glucose dependence of insulin secretion (46, 47), many properties of
their membrane-signaling and of the Ca2+-dependent
insulin-secretion process closely resemble those in ß-cells. RINm5F
cells respond to insulin secretagogues (such as glucose,
glyceraldehyde, alanine, and sulfonylureas) with membrane
depolarization, resulting in LTCC activation and spike activity (48).
LTCCs in RINM5F cells possess pharmacological and electrophysiological
properties, e.g. single-channel conductance and gating
kinetics, indistinguishable from those in ß-cells. This also holds
true for their modulation by protein kinases A and C (1, 8, 49).
Therefore, these cells should serve as a suitable biochemical model of
(neuro)endocrine LTCCs coupled to insulin secretion.
Differential Phosphorylation of Two Size Forms of
1C in RINm5F
Cells
Agents that increase cytosolic cAMP enhance stimulus-secretion
coupling in ß-cells. This is attributed to cA-PK-mediated stimulation
of Ca2+-influx through LTCCs as well as to direct effects
on the secretory machinery itself (1, 43). Ca2+ influx is
increased by potentiation of LTCC activity due to a
phosphorylation-induced slowing of channel inactivation (43).
cA-PK-mediated LTCC modulation in brain, skeletal muscle, and cardiac
muscle could be mediated by direct phoshorylation of the C-terminal
portions of the channels
1-subunits (32, 41, 50, 51, 52, 53). Using
sequence-directed
1-subunit antibodies, we demonstrate the existence
of at least two different classes of
1-polypeptides. One class was
unequivocally identified as
1C. The apparent molecular mass of
1C
in immunoblots of 199 kDa indicated that it does not represent its
full-length form. This was confirmed by phosphorylation of
immunopurified fractions disclosing a long form that serves as the
substrate for cA-PK in vitro. This form must therefore
contain the C-terminal site phosphorylated by cA-PK [serine 1928 of
the class C sequence (32, 41, 53)]. Such posttranslational proteolytic
processing of Ca2+ channel
1 C-termini has also been
described in skeletal muscle (50), brain (51, 52), and heart (32, 53).
Here we demonstrate that it also occurs in a (neuro)endocrine cell,
indicating that the protease responsible for this cleavage is also
expressed in RINm5F cells. It was proposed that this cleavage would
allow the differential modulation of the two size forms of
1 by
cA-PK-dependent phosphorylation (52, 54). This could be especially
important if the two forms are targeted to different regions within the
plasma membrane. Specific targeting of DHP-sensitive Ca2+
channels to specific plasmalemmal compartments has not only been
reported for skeletal muscle myocytes (55) and neurons (34), but also,
more recently, for insulin-secreting ß-cells (56). In these cells
LTCCs are preferentially located on the secretory vesicle-containing
side of the plasma membrane, where their activity is tightly coupled to
insulin secretion (56).
It is also possible that C-terminal truncation and phosphorylation
affect the interaction of the
1-subunit with ß-,
2-
-, or G
protein subunits, thereby modulating channel function. Further
biochemical studies are required to address this important
question.
1C-, ß1b-, and ß3-Subunits Are Major Constituents of
(+)-[3H]isradipine-Labeled LTCC in RINm5F
Cells
Immunostaining of channels partially purified by lectin affinity
chromatography revealed that
1C represents only a fraction of the
total
1-subunit immunoreactivity as detected in RINm5F cells with
our nonselective antibody (anti-
1). These
1-subunits most likely
represent non-LTCC
1-subunits, which are also expressed in these
cells (8, 33), or translation products of the class D gene.
1D
Transcripts have been found to be abundant in insulin-secreting cells
(21, 22, 23, 24). Similar to
1C and
1S,
1D-subunits were shown to
mediate DHP agonist- and antagonist-sensitive L-type Ca2+
currents after heterologous expression (24, 57) and are believed to
bind (+)-[3H]isradipine with similar affinity as
1C
(51). Whether about one half of
(+)-[3H]isradipine-binding activity not recognized by
anti-
1C in our experiments is associated with an LTCC complex formed
by
1D or is sterically not accessible for our anti-
1C antibodies
remains to be determined. In rat pancreatic islets,
1C and
1D
represent about one and two thirds, respectively, of L-type
1-transcripts (21). This is similar to our biochemical finding of
approximately 50%
1C, suggesting that the relative expression of
1C vs.
1D is similar in rat RINm5F cells and rat
pancreatic islets. This again argues for the validity of these cells as
a biochemical model for ß-cell LTCCs.
We also provide evidence that all four known ß-subunit isoforms
participate in the formation of LTCCs in brain to a different extent
(Fig. 3
, D and E). However, in RINm5F cells we only found a substantial
association of ß1b- or ß3-subunits with channel complexes. This is
in good agreement with the preliminary finding that ß3- and
ß1-transcripts can be detected in insulin-secreting cells at higher
levels (25) than ß2 (25, 26) and ß4 (25, 38). Although the
possibility exists that the relative abundance of the two isoforms
differs in RINm5F cells from that in pancreatic ß-cells, our data
clearly emphasize that future studies should focus on the role of these
two subunits for LTCC function in pancreatic ß-cells. It will be
important to determine whether these isoforms confer different
biophysical or pharmacological properties to coexpressed
1C- or
1D-subunits in heterologous systems.
We would like to point out that our data cannot predict to which extent
LTCC complexes formed by
1C-, ß1b-, or ß3-subunits contribute to
voltage-gated Ca2+ entry in RINm5F or ß-cells. However,
they provide important biochemical information that will help to
address this question, e.g. by employing
antisense-techniques (58) in these cells.
Based on our data, ß1b- and ß3-subunits will be the ß-subunits of
choice for the generation of cell lines stably expressing
(neuro)endocrine LTCCs. Such cell lines could prove to be important
tools for the development of drugs selectively modulating LTCCs in
neuroendocrine cells such as pancreatic ß-cells. In a recent report,
1D-subunits were expressed together with ß2- subunits cloned from
RINm5F cells in an attempt to establish such a cell line (24). However,
based on our data, the physiological significance of this subunit
combination remains questionable.
We cannot rule out that an even higher percentage of LTCCs are
associated with ß3- and ß1b-subunits in RINm5F cell and brain. In
contrast to our nonselective anti-ß-antibody, our isoform-specific
antibodies were raised against C-terminal portions of these subunits
for several reasons. This region is not conserved among subunit
isoforms, seems not to undergo alternative splicing [as does, for
example, the N-terminal region (37)] and is more likely to be
accessible for the antibody under nondenaturating conditions. However,
our antibodies would not detect ß-subunits with a proteolytically
degraded C terminus. Although we do not have evidence for major
proteolytic degradation of ß3- and ß1b-subunits (Fig. 4
, A and B)
in immunoblots, this possibility cannot be completely excluded.
Taken together, we demonstrate that
1C-, ß3-, and ß1b-subunits
are major constituents of (+)-[3H]isradipine-labeled
LTCCs in RINm5F cells. The full length form of
1C serves as an
in vitro substrate for cAMP-dependent phosphorylation.
Backphosphorylation experiments of these subunits in RINm5F cells with
different protein kinases should provide a powerful biochemical tool
with which to study the effects of hormones, drugs, and glucose
metabolism on Ca2+ channel modulation in insulin-secreting
cells.
 |
MATERIALS AND METHODS
|
---|
Materials
Materials were obtained from the following sources: ATP, sheep
anti-rabbit-IgG (conjugated to alkaline phosphatase), catalytic subunit
of cA-PK, protein A-Sepharose, protease inhibitors, and
N-acetyl-glucosamine from Sigma (St. Louis, MO);
CNBr-activated Sepharose 4B from Pharmacia (Vienna, Austria); digitonin
from Biosynth AG (Basel, Switzerland); [
32P]ATP from
Chemomedica (Vienna, Austria); (+)-[3H]isradipine (80
Ci/mmol) from New England Nuclear (Vienna, Austria); okadaic acid and
soybean trypsin inhibitor from Boehringer (Vienna, Austria); X-Omat AR5
films from Kodak (Rochester, NY); Immobilon-P transfer membranes from
Millipore (Bedford, MA); and prestained molecular weight markers from
Bio-Rad (Vienna, Austria) and GIBCO BRL (Vienna, Austria). Unlabeled
isradipine was a gift from Sandoz AG (Basel, Switzerland).
Cell Culture
The insulin-secreting RINm5F insulinoma cell line was originally
provided by C.B. Wollheim (Geneva). Cell culture was carried out as
described in detail elsewhere (11).
Membrane Preparation
RINm5F cells were resuspended in 50 mM Tris-HCl
supplemented with 2 mM MgCl2, 2 mM
EDTA, 0.5 mM phenylmethylsulfonylfluoride (PMSF), and 1
µg/ml soybean trypsin inhibitor, pH 7.6, and lysed by ten strokes in
a Dounce homogenizer. Membranes were obtained by sequential
centrifugation at 700 x g and 40,000 x
g for 15 and 35 min (4 C), respectively. The 40,000 x
g pellet was resuspended in binding buffer (50
mM Tris-HCl, pH 7.4, 0.1 mM PMSF) at a final
protein concentration of 514 mg of protein/ml). Rabbit and guinea pig
cardiac and brain membranes were prepared as described (59).
Sequence-Directed Antibody Production and Immunoblotting
Sequence-directed antibodies were raised by immunization of
New-Zealand White rabbits with synthetic peptides coupled to BSA as
described (35). Antibody anti-
1 was raised against a synthetic
peptide corresponding to residues 13821400 of the skeletal muscle
1-subunit (35). This sequence is highly conserved among all
Ca2+ channel
1-subunits. Antibody anti-
1C is directed
against a synthetic peptide corresponding to residues 818835 of the
1C-subunit (45). Antibody anti-
2 was raised against a synthetic
peptide corresponding to residues 721738 of the skeletal muscle
2-subunit (60). Non-isoform-specific anti-ß-subunit antibodies
were generated against peptides corresponding to residues 6179 of
ß1a (anti-ß). This sequence is highly conserved among all four
ß-subunit genes (38). Isoform-selective antibodies were generated
against the following residues: ß1b, 516530; ß2, 595604; ß3,
470483; ß4, 460474 (16, 38). Immunoblotting was carried out as
described previously (61) employing affinity-purified (35) antibodies.
Nonspecific staining was assessed by preincubation of antibodies with
the antigenic peptide (1 µM). Affinity-purification was
carried out as described (35).
Lectin Sepharose Affinity Chromatography
Ca2+ channels in digitonin extracts of the RINm5F
cell membrane fraction were partially purified by WGA-Sepharose
chromatography as described previously (62). In typical experiments
about 10 mg of membrane protein were prelabeled with a saturating
concentration of (+)-[3H]isradipine (23 nM)
in 510 ml of 50 mM Tris-HCl, pH 7.4, 1 mM
CaCl2, 0.1 mM PMSF (37 C, 45 min). Membranes
were collected by centrifugation (40,000 x g) and
resuspended in the same buffer containing 0.15 M NaCl and
1% (wt/vol) digitonin. After incubation for 30 min on ice, nonsoluble
material was removed by centrifugation (30 min, 100 000 x
g). Twenty to 30% (range, n = 6) of DHP-binding
activity was recovered in the extract together with 5060% of the
total membrane protein. Solubilized binding activity was purified on 2
ml WGA-Sepharose (10 mg of wheat germ lectin immobilized per 1 ml of
Sepharose 4B Cl) as described for skeletal muscle Ca2+
channels.
Double Immunoprecipitation of Phosphorylated
1-Subunits
Immunoprecipitation and phosphorylation of RINm5F
Ca2+ channels were carried out as described by Hell
et al. (63) with minor modifications. Briefly, 0.2 mg
membrane protein was solubilized in 0.250.4 ml RIA buffer (20
mM Tris-HCl, 150 mM NaCl, 0.05% BSA; pH 7.4,
1% [vol/vol] Triton X-100 for 30 min on ice followed by
centrifugation for 5 min at 10,000 x g at 4 C.
Solubilized protein was preadsorbed (30 min at 4 C) to a mixture of
protein A-Sepharose (5 µl) and Sepharose CL-4B (75 µl). After
centrifugation for 30 sec, 200 µl of the supernatant were added to a
solution (10 µl-50 µl) of affinity-purified antibodies in TBS
(anti-
1C or the control antibodies anti-
2 or anti-pgp-389, see
below) and incubated for 2 h at 4 C. Washed protein A-Sepharose
(
20 µl swollen gel in RIA buffer) was added to the samples and
further incubated for 2 h at 4 C. The protein A Sepharose was then
washed three times with 1.25 ml RIA buffer and once with
phosphorylation buffer (20 mM HEPES, 4 mM MgCl,
0.4 mM EGTA, 0.04% Triton X-100, 0.1 mM PMSF,
1 µM pepstatin A, 12.5 mM NaF, and 20
mM ß-glycerolphosphate). Antibody-bound channel was
phosphorylated (30 C for 20 min) in the presence of 3 µM
[
32]ATP (specific activity, 100500 dpm/fmol) and
cA-PK (10 U). The resin was washed three times with RIA buffer
containing 20 mM EDTA, 20 mM
ß-glycerolphosphate, and 12.5 mM NaF (RIA-P buffer) and
two times with RIA buffer followed by a wash in TBS. Precipitated
protein was eluted from the first-step antibody by denaturation in 1%
(wt/vol) SDS, 50 mM Tris-HCl, pH 7.4, 5 mM
dithiotreitol, 20 mM ß-glycerolphosphate, 0.1
mM PMSF, and 1 µM pepstatin A (30 min, 56 C).
The eluate was diluted 10-fold in RIA-P buffer before the second-step
antibody (anti-
1) was added. After incubation for 2 h (4 C) the
antibody-antigen complex was again recovered on Protein A-Sepharose as
above. The resulting protein A-Sepharose pellet was washed three times
with RIA buffer and once with TBS. SDS-PAGE sample buffer was added to
the washed pellet and subjected to SDS-PAGE (SDS gel electrophoresis,
8% polyacrylamide) under reducing conditions (sample buffer containing
10 mM dithiothreitol).
Immunoprecipitation of
(+)-[3H]Isradipine-Prelabeled Channels
Ten to 20 mg of RINm5F membrane protein were prelabeled with
(+)-[3H]isradipine (1.511 nM) in 510 ml
of 50 mM Tris-HCl, pH 7.4, 1 mM
CaCl2, 0.1 mM PMSF (37 C, 45 min). Membranes
were collected by centrifugation, and digitonin extracts were prepared
after resuspension in 510 ml solubilization buffer as described
above. Aliquots (0.30.75 ml) of the extract were added to
affinity-purified antibodies immobilized on Protein A-Sepharose
(0.030.05 ml) equilibrated in TBS containing 0.1% digitonin. After
incubation for 34 h at 4 C, unbound radioactivity was removed by
extensive washing (4 x 1.5 ml) with ice-cold TBS containing 0.1%
digitonin. Bound radioactivity was determined by liquid scintillation
counting of the washed resin. An affinity-purified, sequence-directed
antibody that efficiently immunoprecipitates p-glycoprotein
[anti-pgp-389 (64)] was used to determine nonspecific
immunoprecipitation.
Note that anti-
1 could not be used for these immunoprecipitation
experiments. It binds to
1-subunits only after denaturation in
detergents such as Triton X-100 or SDS but does not recognize
digitonin-solubilized Ca2+ channels with high affinity
(35).
The amount of precipitable (+)-[3H]isradipine binding was
determined on ice as follows: 0.10.3 ml digitonin extract was added
to 0.1 ml of an ice-cold solution of 50 mM Tris-HCl, pH
7.4, containing 5 mg BSA/ml and 5 mg
-globulin/ml. The mixture was
rapidly diluted in 10 mM Tris-HCl, pH 7.4, 10% (wt/vol)
polyethylene glycol 6000, and 10 mM MgCl2 and
incubated on ice for 2 min. Precipitable
(+)-[3H]isradipine-binding activity was determined by
filtration over GF/C Whatman glass fiber filters as described
previously (59) followed by liquid scintillation counting. To measure
nonspecific (+)-[3H]isradipine binding, prelabeling was
carried out in the presence of 1 µM (±)-isradipine.
Nonspecifically bound (+)-[3H]isradipine was not
immunoprecipitated by our antibodies (n = 2).
Enrichment of ß-Subunits by Affinity Chromatography on
GST-AIDA-Sepharose
The
1-interaction domain of the
1A-subunit (AIDA) was
overexpressed as a fusion protein (GST-AIDA) with
glutathione-S-transferase (GST) in Escherichia coli and
purified and coupled to glutathione-Sepharose beads as described
previously (17). Ten milligrams of membrane protein were solubilized
(30 min, 4 C) in 4.5 ml buffer A (50 mM Tris-HCl, pH 7.4,
0.1 mM benzamidine, 1.0 mM iodoacetamide, 0.1
mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptine, 1
µM pepstatin A, 20 µg/ml calpain inhibitors I, and II)
containing 1% (wt/vol)
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate and 1
M NaCl. Insoluble material was removed by centrifugation
(45,000 x g, 30 min, 4 C), the supernatant was diluted
10-fold with buffer A, and 4 ml were subjected to purification on
GST-AIDA Sepharose beads (0.03 ml, 0.01 mg GST-AIDA) and SDS-PAGE as
described (17).
SDS-PAGE
Membrane proteins were separated on 710% straight
polyacrylamide gels in a Bio-Rad Minigel apparatus under reducing
conditions (10 mM dithiothreitol included in the sample
buffer). Apparent molecular masses of Ca2+ channel subunits
were determined by linear regression analysis from standard curves
obtained with prestained marker proteins separated on the same
gels.
Protein Assay
Protein was quantified according to the method of Bradford (65)
using BSA as a standard.
Statistics
Except when stated otherwise, data are given as means ±
SE from the indicated number of experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank D. Ostler, E. Penz, A. Lemmermöhle, D. Reimer,
and E. Emberger for expert technical assistence, Drs. M. Grabner and J.
Mitterdorfer for helpful comments, and Professor H. Glossmann for
providing unlabeled Ca2+ antagonists and continuous
support.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jörg Striessnig, Institut für Biochemische Pharmakologie, Peter-Mayrstraße 1, A-6020 Innsbruck, Austria.
This work was supported by the Fonds zur Förderung der
Wissenschaftlichen Forschung (S6602 to J.S.), a research grant from the
Deutsche Diabetes-Gesellschaft, the Deutsche Froschungsgemeinschaft (to
H.H.), and research fellowships from the Graduiertenförderung des
Landes Baden-Württemberg (to U.K. and M.R.). T.N.C was supported
by a Fullbright Fellowship.
Received for publication February 16, 1996.
Revision received January 13, 1997.
Accepted for publication February 3, 1997.
 |
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