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1D) Calcium Channel Subunit from an Insulin-Secreting Cell Line
Institut für Pharmakologie (A.S., T.D.P., B.N.) Freie
Universität Berlin 14195 Berlin, Germany
Department
of Pharmacology University College London (A.C.D.) WC1E 6BT,
United Kingdom
Abteilung für Pharmakologie und
Toxikologie (B.N.) Universität Ulm 89081 Ulm,
Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1-subunits in
pancreatic ß-cells is of the neuroendocrine subtype
(CaV1.3 or 1D). Here
we studied the biophysical properties and receptor regulation of a
CaV1.3 subunit previously cloned from HIT-T15
cells. In doing so, we compared this neuroendocrine
CaV1.3 channel with the cardiac L-type channel
CaV1.2a (or 1C-a)
after expression together with 2
- and
ß3-subunits in Xenopus oocytes.
Both the current voltage relation and voltage dependence of
inactivation for the neuroendocrine CaV1.3
channel were shifted to more negative potentials compared with the
cardiac CaV1.2 channel. In addition, the
CaV1.3 channel activated and inactivated more
rapidly than the CaV1.2a channel. Both subtypes
showed a similar sensitivity to the dihydropyridine
(+)isradipine. More interestingly, the CaV1.3
channels were found to be stimulated by ligand-bound
Gi/Go-coupled GPCRs
whereas a neuronal CaV2.2 (or
1B) channel was inhibited. The observed
receptor-induced stimulation of CaV1.3 channels
could be mimicked by phorbol-12-myristate-13-acetate and was sensitive
to inhibitors of protein kinases, but not to the
phosphoinositol-3-kinase-inhibitor wortmannin, pointing to
serine/threonine kinase-dependent regulation. Taken together, we
describe a neuroendocrine L-type CaV1.3 calcium
channel that is stimulated by
Gi/Go-coupled GPCRs and
differs significantly in distinct biophysical characteristics from the
cardiac subtype (CaV1.2a), suggesting that the
channels have different roles in native cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using molecular biological approaches, various pore-forming calcium
channel 1-subunits
(CaV1.2, CaV1.3,
CaV2.1, CaV2.2,
CaV2.3, and CaV3.1) have
been identified in insulin-secreting cells and pancreatic tissue
(1, 2, 3, 4, 5). In electrophysiological studies, channel currents with the
pharmacological and functional properties known for these subunits have
also been reported. Thus, high-voltage-activated (HVA), dihydropyridine
(DHP)-sensitive channels (6), corresponding to the
CaV1.2 or CaV1.3 subunits,
-conotoxin GVIA-sensitive channels (7, 8), corresponding to the
CaV2.2 subunit, and -agatoxin IVA-sensitive
channels (5), corresponding to the CaV2.1
subunit, have been described. Low-voltage-activated currents,
which would correspond to the CaV3.1 subunit that
has been found in the INS-1 cell line, have also been observed in
several insulin-secreting cell types (4).
Taken together, insulin-secreting cells express multiple voltage-gated
calcium channel subtypes, making it difficult to clarify the properties
of a certain channel type in these cells. A common approach to address
this problem is the use of cellular systems overexpressing the proteins
of interest. This approach has already been extensively used to study
the properties of channels formed by most of the calcium channel
1-subunits [e.g.
CaV1.2 (9, 10), CaV2.1
(11), CaV2.2 (9, 12, 13),
CaV2.3 (11, 14); CaV3.1
(15, 16)].
In the case of the CaV1.3 subunit, however, which
is thought to play a major role in the regulation of insulin secretion,
few data are available (17, 18), although several splice variants were
cloned (17, 18, 19, 20). This subunit, in combination with the auxiliary
subunits 2 and ß, forms an HVA-calcium
channel of the L-type (for review see Refs. 21, 22, 23). Its occurrence is
not restricted to the pancreas. Isoforms of the
CaV1.3 subunit have been identified by RT-PCR,
antibodies, or Northern blot analysis in brain, heart, kidney, adrenal
gland, and osteoblasts (24, 25, 26). CaV1.3 is
thought to comprise the major fraction of L-type calcium current in
neurosecretory and neuronal cells (Refs. 22, 27 but see also Ref.
25).
In pancreatic ß-cells the majority of L-type calcium channel mRNA
codes for CaV1.3 subunits (2, 28). So far, there
is only a single brief report on the functional expression of a
CaV1.3 channel cloned from an insulin secreting
cell line [RINm5F (17)]. Hence, data on the functional properties and
regulation of these neuroendocrine CaV1.3-derived
pore-forming calcium channel subunits are still lacking. This prompted
us to study the CaV1.3 isoform cloned from the
insulin-secreting HIT-T15 cell line (19), after coexpression with the
2 and ß3 subunits
in Xenopus oocytes. Here, we provide the first description
of the biophysical properties of this isoform and the first description
of receptor regulation of a heterologously expressed pancreatic
CaV1.3 channel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 subunits encoding the
neuroendocrine (CaV1.3), cardiac
(CaV1.2a), or neuronal
(CaV2.2) calcium channel together with the
2 and ß3 subunits
into oocytes. The resulting Ba2+ inward current
amplitudes in subsequent voltage-clamp measurements were highly
variable (Fig. 1A
). Differences were
considerable between individual donor toads, but much less within a
batch of oocytes from the same animal. Uninjected oocytes showed
either no detectable native calcium channel current, or inward currents
with amplitudes of up to 50 nA. In oocyte batches from most donor
animals, endogenous oocyte calcium channel currents were enhanced by
exogenous 2 and ß subunits, as described
previously (29). The resulting currents were often of considerable
amplitude (see Fig. 1A), in agreement with one previous study (29), but
in contrast to others (18, 30). Hereafter, the
ß/2-stimulated endogenous oocyte calcium
channel is referred to as the endogenous calcium channel. To assess the
effects on endogenous channels, all types of experiments on inward
Ba2+ currents were also performed in cells
injected with cRNA for the 2 and ß
subunits. To minimize the contribution of currents through the
endogenous channel, the amplitudes of currents through these channels
were compared with those from cells injected with cRNA for the
respective complete exogenous channel. Only those oocyte batches were
used, in which a very large relative proportion (at least 80%) of
total current was contributed by the exogenous channel subtype. Up to
20% of endogenous current was present in some cases when examining the
CaV1.3 channel, since the amplitude of currents
in oocytes expressing this channel was in general smaller than that in
those expressing the cardiac CaV1.2a or neuronal
CaV2.2 subunits (compare Fig. 1A). These latter
two 1 subunits produced large currents, which
were usually measurable on the second day after cRNA injection. In
contrast, expression of the neuroendocrine CaV1.3
calcium channel exceeded expression of the endogenous channel to a
sufficient extent for measurement only after 4–6 days.
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2 and ß (n = 8), the neuroendocrine
CaV1.3 subunit did not produce inward currents
exceeding those of noninjected oocytes, in contrast to the
CaV1.2a subunit (our unpublished data and
Ref. 31). Currents of larger amplitude than those in control cells were
also not observed when only 2 was expressed
together with CaV1.3. In contrast, when the
ß3 subunit was coexpressed with
CaV1.3 without 2, the
resulting current amplitudes were 22–50% of the corresponding
CaV1.3/ß3/2
currents. In one batch of oocytes that did not display measurable
endogenous calcium channel currents after injection of exogenous
auxiliary subunits, the following maximum inward current amplitudes
were obtained: CaV1.3: 0 nA (n = 4),
CaV1.3/ß3: -152 ±
29 nA (n = 6),
CaV1.3/ß3/2:
-586 ± 141 nA (n = 6).
Activation of Ba2+ Currents
Analysis of the current-voltage (I-V) relationships for the
neuroendocrine CaV1.3 and the cardiac
CaV1.2a channels (Fig. 1B
) revealed significant
differences with respect to the half-maximal activation parameters
(V1/2; P <
0.05), which were -9.6 ± 1.2 mV (n =
17) and -6.1 ± 1.2 mV (n = 17), but not for the slope
factors (k), which were 7.5 ± 0.2 mV and 7.3 ± 0.4 mV,
respectively. The maximum inward current for the neuroendocrine
CaV1.3 channel occurred at more negative
potentials (2.7 ± 1.1 mV; n = 14) than for the cardiac
CaV1.2a channel (6.4 ± 1.1 mV;
P < 0.05). The I-V relation of the endogenous calcium
channel showed a V1/2 that was significantly
(P < 0.001) shifted to depolarized potentials compared
with the CaV1.3 and the
CaV1.2a channels. Half-maximal activation
occurred at 9.4 ± 1.1 mV, and the slope factor (k) was estimated
to be 7.5 ± 0.4 mV. The maximum inward current was at 18.8
± 1.0 mV, a value comparable to those published previously (10, 18, 31).
It is obvious from the current traces in Fig. 1C that the
activation of the cardiac CaV1.2a channel is
slower than that of the neuroendocrine CaV1.3 or
endogenous counterparts. The rising phase of currents elicited by
depolarizing voltage steps was quantified by determining the time
necessary to reach 90% of peak current [90% time-to-peak (ttp)]
in each case (Fig. 2A). The cardiac
CaV1.2a calcium channel showed a significantly
(P < 0.05) slower activation compared with the
neuroendocrine CaV1.3 and the endogenous channel
over the voltage range between -10 and +20 mV. The activation times
for the neuroendocrine CaV1.3 and the endogenous
oocyte channels were only significantly different (P <
0.05) at -10 mV. In addition, a voltage dependence of the values for
90% ttp was observed for all three channels with values decreasing
with increasing depolarization. This decrease with potential was,
however, only significant (P < 0.05) for the
neuroendocrine CaV1.3 and the cardiac
CaV1.2a channels, when the values of 90% ttp at
the different voltages were compared for each individual subtype.
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and 2B, it is clear that the
rate of inactivation of calcium channel currents differs for the
neuroendocrine CaV1.3, cardiac
CaV1.2a, and endogenous channel subtypes. When
the current decay during longer depolarizing voltage steps was analyzed
in detail, it was found to be best fitted by a monoexponential function
(eq 4) when pulses of 1,500 msec were used. Currents during longer
pulses (5 sec) were better fitted by a biexponential function (eq 5,
values in Table 1). The neuroendocrine
CaV1.3 and the cardiac
CaV1.2a channels differed significantly, both in
their 
1 (P < 0.005) and
2 (P < 0.005) values and in
the ratio of the amplitudes of the fast and the slow current components
(A1/A2, P
< 0.001). The endogenous channel inactivated most rapidly, but
differed significantly (P < 0.05) from the
neuroendocrine CaV1.3 channel only in the
1 value for the fast current component.
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), was 546 ± 23 msec (n = 17) for the neuroendocrine
CaV1.3 and 972 ± 126 msec (n = 9) for
the cardiac CaV1.2a calcium channel, values that
were significantly different (P < 0.001; see also Fig. 2B). Since endogenous oocyte
Ca2+-activated Cl-
conductances may obscure the measurement of inactivation of calcium
channels, additional experiments were performed on oocytes with the
calcium chelator BAPTA
[1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic
acid, sodium] present inside the cell. The currents during 1,500-msec
pulses to the maximum of the individual I-V curve fitted with a
monoexponential function, was 530 ± 15 msec (n = 26) for
the neuroendocrine CaV1.3 and 966 ± 88 msec
(n = 14) for the cardiac CaV1.2a calcium
channel, values that were significantly different (P <
0.001). Hence, the rate of inactivation of
CaV1.2a channels was significantly different from
inactivation of CaV1.3 channels, both in the
presence and absence of the intracellular Ca2+
chelator BAPTA.
Potential Dependence of Ba2+ Current
Inactivation
The steady-state inactivation was measured with a two-step
protocol. The inactivation curves are shown in Fig. 2C. Their analysis
indicated that the cardiac CaV1.2a and the
neuroendocrine CaV1.3 channels differ
significantly (P < 0.001) in their half-maximal
inactivation, with respective V1/2 values of
-22.7 ± 1.7 (n = 20) and -36.1 ± 0.9 mV (n =
24). Values for the endogenous calcium channel
(V1/2 = -35.0 ± 3.5 mV; n = 7)
differed significantly from both other channels (P =
0.001). The slope factor (k) was almost identical for all three
channels (neuroendocrine CaV1.3: 12.7 ± 0.3
mV; cardiac CaV1.2a: 14.0 ± 0.7 mV; and
endogenous: 14.4 ± 1.2 mV).
In addition, we analyzed steady-state inactivation in the presence of intracellular BAPTA. The results also showed a significant difference (P < 0.001) between the cardiac CaV1.2a channel (V1/2 = -20.5 ± 2.3 mV; n = 15) and the neuroendocrine CaV1.3 channel (V1/2 = -30.9 ± 1.2 mV; n = 17).
Current Characterization by Blocking Agents
HVA calcium currents in native ß-cells have been shown to be
blocked completely by 200 µM Cd2+
(8). Addition of 200 µM Cd2+ to the
bath solution abolished currents through the
CaV1.3/ß3/2
(n = 6) as well as the
CaV1.2a/ß3/2
(n = 4) calcium channels.
An important distinguishing feature for L-type calcium channels is
their sensitivity to DHPs. We therefore compared the DHP sensitivity of
the neuroendocrine CaV1.3, cardiac
CaV1.2a, and endogenous channels. Using
(+)isradipine (Fig. 3), we found a 50%
inhibition of the DHP-sensitive fraction of the current at a
concentration of 43 nM for the cardiac
CaV1.2a channel and a similar value of 57
nM for the neuroendocrine CaV1.3
channel. The endogenous current was not inhibited by (+)isradipine at
concentrations up to 3 µM (n = 7), as also shown in
other studies (31, 32).
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1A,B,E-subunits
by Gß
-complexes (12, 35, 36). Furthermore, the neuroendocrine
CaV1.3 calcium channel subtype, in particular,
has been proposed to be affected by this inhibition (for review see
Ref. 22). This prompted us to examine the regulation of the
neuroendocrine CaV1.3 channel from HIT-T15 cells
in oocytes coinjected with the
Gi/Go-coupled µ-opioid
receptor. In our hands, this receptor was able to couple to endogenous
phospholipase C in oocytes, as indicated by the stimulation of
calcium-dependent chloride currents (n = 14) after receptor
activation with the selective µ-opioid receptor agonist DAMGO
([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin)
in oocytes bathed in modified Ringer. Furthermore, we have ascertained
that the µ-opioid receptor used in this study couples only to
pertussis toxin-sensitive G proteins of the
Gi-subfamily regardless of which agonist was used
(37). Moreover, as a control for G protein-mediated inhibition in this
system, we tested the neuronal (N-type) CaV2.2
(or 1B) calcium channel. This channel is the
classical example of a voltage-dependent calcium channel inhibited by
PT-sensitive G proteins (38, 39). As depicted in Fig. 4, application of DAMGO to oocytes
coexpressing µ-opioid receptors and CaV2.2
channels resulted in a rapid inhibition of inward currents. The
concentration of DAMGO necessary to maximally inhibit this channel was
determined from the dose-response relation, which revealed an
IC50 of 1.1 nM and maximal
inhibition with concentrations above 100 nM
(n = 3–5). This maximal inhibition was approximately 60%, in
accordance with previous studies of this channel when expressed with
calcium channel ß subunits (9, 40). In contrast to the neuronal
CaV2.2, the neuroendocrine
CaV1.3 channel was stimulated by DAMGO at a
maximally effective concentration (1 µM). The
effect started within a few seconds of agonist application, reached a
maximum increase of 406 ± 49 nA (28 ± 2%, n = 20)
within 2 min, and was reversible upon washout of the agonist (see Fig. 4). To exclude nonspecific, receptor-independent effects, DAMGO was
applied at the same concentration to cells injected with cRNA for the
neuroendocrine CaV1.3 channel, but not for the
µ-opioid receptor. No stimulation of inward current was observed
(n = 5) under these conditions or after the application of bath
solution without DAMGO to cells expressing the µ-opioid receptor and
neuroendocrine CaV1.3 channel (n = 3). Since
it was shown by Zamponi et al. (40) that G protein-mediated
inhibition can be impaired as a result of channel phosphorylation by
protein kinase C (PKC), oocytes were incubated with H7 at a high
concentration (100 µM) to inhibit
serine/threonine kinase-dependent phosphorylation as completely as
possible (Fig. 4B). After 5 min (n = 3) or 30 min (n = 2) of
preincubation with H7, the increase in current through the
neuroendocrine CaV1.3 channel after opioid
receptor activation was prevented. However, under these conditions
DAMGO still failed to induce calcium channel inhibition (see Fig. 4B).
We therefore concluded that DAMGO-induced neuroendocrine
CaV1.3 calcium channel stimulation involved
protein phosphorylation. Stimulation of calcium channel currents in
neuroendocrine cells resulting from phosphorylation by protein kinase A
(PKA) and PKC has been reported in several studies (41, 42). Since PKA
can also be inhibited by H7 (43), we tested the effect of PKA
inhibition on receptor-stimulated channel activity. The response of the
neuroendocrine CaV1.3 calcium channel to receptor
activation in oocytes injected with the PKA-inhibitor Rp-cAMPS
(adenosine 3',5'-cyclic phosphorothiolate-Rp) [to calculated final
concentrations of 50 µM (n = 4) and 400
µM (n = 5), 15–20 min before
experiments], did not significantly differ from that in control cells
without Rp-cAMPS (n = 9). Since L-type calcium channels are known
to be stimulated by G protein-coupled receptors (GPCRs) in a
phosphoinositol-3-kinase (PI3K)- and/or PKC-dependent manner, we tested
the effects of the PI3K-inhibitor wortmannin and the PKC-inhibitor
bisindolylmaleimide I on calcium current stimulation. Incubation of
oocytes with 150 nM and 500
nM wortmannin in the extracellular solution for
20 min did not result in a significant reduction of the DAMGO-induced
effect (n = 5). In contrast, preincubation of oocytes with
bisindolylmaleimide I (100 nM, 1 h) resulted
in a significant reduction of DAMGO-induced calcium current stimulation
(P < 0.005; Fig. 4B). Furthermore, we used the phorbol
ester phorbol myristate acetate (PMA) to directly activate PKC and
obviate potential complications due to overlapping inhibitory effects
via Gi/o proteins and stimulatory effects via PKC
on calcium channel activity. In line with the results obtained with
DAMGO, acute application of PMA (100 nM; n =
12) increased the Ba2+ inward current (107.5
± 13.4 nA, n = 12) significantly within 2.5 min when compared
with results from experiments with vehicle only (46.7 ± 11.1 nA,
n = 9) in cells overexpressing the neuroendocrine
CaV1.3 channel (P < 0.005).
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). After agonist application, the
increase in currents through the endogenous channel started with a
delay, reaching a maximum usually during washout of the agonist (see
Fig. 4). Since no current stimulation occurred in the presence of H7,
stimulation of the endogenous channel probably results from protein
phosphorylation. This observation is also in agreement with those that
showed that the amplitude of currents through the endogenous oocyte
calcium channel was increased by PMA (Ref. 32 and our unpublished
data). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2 subunits (19). Channels of this subunit
combination are likely to occur in native endocrine cells since
CaV1.3, ß2, and
ß3 have been found to coincide in RINm5F cells
(17). Furthermore, studies using in situ hybridization and
immunostaining found a colocalization of CaV1.3
and ß3 in several brain areas (44).
Notably, currents through this neuroendocrine channel were not observed
upon expression of the CaV1.3 subunit alone or in
combination with 2. Hence, its association
with at least ß subunits seem to be essential for functional
expression, as also reported for two other CaV1.3
isoforms (17, 18). Nevertheless, upon coexpression of
2 with the
CaV1.3/ß3 subunits, the
amplitude of neuroendocrine CaV1.3 channel
currents was increased by 2- to 5-fold in the present study. Similar
effects of the 2 subunit on the
CaV2.1/ß combination were reported by de Waard
and Campbell (30), who found a 2.5-fold current increase. An even more
pronounced effect (
15-fold stimulation) was also reported (10), when
CaV1.2a/ß/2
channels were compared with CaV1.2a/ß
channels.
The neuroendocrine CaV1.3 channel examined here
yielded, on average, relatively low current amplitudes as compared with
channels formed by the CaV2.2 and
CaV1.2a subunits in the same expression system.
Furthermore, the time interval after cRNA injection that was necessary
to obtain currents of sufficient amplitude for kinetic analysis was
longer than that required for the other 1
subunits. Reasons for this observation may be inherent to the
CaV1.3 subunit itself. As suggested by Hofmann
et al. (23), one reason may be the need for additional,
possibly still unidentified, auxiliary subunits. Another possible cause
could be the necessity for specific interactions with additional
proteins, as described for syntaxin and the
CaV1.3a subunit in pancreatic ß-cells (27).
When the I-V curves for the neuroendocrine CaV1.3
channel used in the present study were analyzed, the maximum inward
currents were found to occur at potentials between 0 and +10 mV. A
similar value of 0 mV was reported by Williams et al. (18)
for a neuronal
CaV1.3/ß2/2
subunit combination expressed in Xenopus oocytes. A
particularly interesting native cell for comparison in this respect is
the rat pinealocyte, which has been shown to contain
CaV1.3 as the sole calcium channel
1 subunit, together with the
ß2 and ß4 subunits. The
minimum of the I-V curve in these cells is also between 0 and 10 mV at
similar Ba2+ concentrations (26). When comparing
the potential dependence of the currents, it should be kept in mind
that the ß subunit type also influences this property of calcium
channels and, thus, the position of the I-V curve (23). The comparison
of the I-V minima, which we performed here, seems to be applicable,
since at least in the case of the CaV2.1 subunit,
ß2 or ß3, the isoform
used in the present study, had a similar influence on the potential
dependence of the currents (30).
During prolonged depolarizations, the neuroendocrine CaV1.3 channel showed slow inactivation with Ba2+ as the charge carrier. Inactivation was, however, on average twice as fast for the neuroendocrine CaV1.3 than for the cardiac CaV1.2a channel. The voltage dependence of inactivation also differed significantly between these two L-type channels, with the neuroendocrine CaV1.3 subtype inactivating at more negative membrane potentials. In mouse pancreatic ß-cells, where the majority of the calcium current has been reported to be DHP-sensitive, inactivation during short voltage pulses was shown to be almost purely Ca2+-dependent (3). Other authors also reported a strong calcium-dependence in mouse pancreatic ß-cells (45), but detected an additional slower voltage-dependent component of HVA current inactivation when using longer depolarizing pulses and Ba2+ as the charge carrier. The inactivation of the neuroendocrine CaV1.3 channel in our study, also using Ba2+ as the charge carrier, could partially reflect this component. Although the Ca2+-dependent component dominates inactivation in ß-cells, slower voltage-dependent inactivation could play a role in the regulation of calcium entry during slow wave depolarization.
Sensitivity to DHPs is a property that has frequently been used to distinguish the role of different calcium channel subtypes in the regulation of insulin secretion and the signal transduction processes in insulin-secreting cells (6, 46, 47). Comparison of the DHP sensitivity of the neuroendocrine CaV1.3 and the cardiac CaV1.2a channel revealed similar IC50 values for (+)isradipine. Therefore, this DHP does not discriminate between these two channel isoforms.
Regulation of L-type calcium channels in insulin-secreting cells,
either directly by heterotrimeric G proteins or through second
messengers, is a matter of intense research (6, 42). For instance,
GPCRs have been reported to inhibit L-type calcium currents in
insulin-secreting RINm5F cells via PT-sensitive G proteins (6, 7, 47). Since heterologously expressed CaV1.2
(1C) calcium channels are not a target for G
protein-dependent inhibition (9, 35), CaV1.3
(1D) isoforms are L-type channel candidates
for inhibition by G proteins. However, in the present study, activation
of PT-sensitive G proteins by µ-opioid receptors did not result in
inhibition of the neuroendocrine calcium channel
(CaV1.3 or 1D) despite
clear inhibition of the neuronal calcium channel
(CaV2.2 or 1B) under
identical experimental conditions. In this context it should be noted
that the CaV2.2 contains the known
Gß-binding motif QXXER present in the intracellular loop between
repeats I and II. Resequencing of the CaV1.3 by
us revealed the presence of a QXXEE-amino acid motif in the loop I-II
sequence, which has been found in various L-type calcium channels
including a neuronal isoform known not to be directly inhibited by
Gß (23, 39, 48).
In contrast, we found that currents through this
CaV1.3 channel were increased after receptor
activation. The stimulation of the CaV1.3 channel
is clearly distinguishable from that of the endogenous channel both in
time course and amplitude. The finding that the specific kinase
inhibitors H7 and bisindolylmaleimide I abolished this receptor-induced
current stimulation is mechanistically relevant and points to the
involvement of a kinase. Stimulation of voltage-dependent calcium
channels by PKC or PKA is well established (23). H7 inhibits both
kinases with IC50 values in a comparable range
whereas bisindolylmaleimide I is a potent inhibitor of PKC (43, 49).
Furthermore, we recently showed that smooth muscle L-type calcium
channels are activated by Gß-subunits via PKC- and PI3K-dependent
pathways (50, 51). However, for GPCR-controlled regulation of the
CaV1.3 channel examined in this study, the
involvement of PI3K is unlikely, since the specific PI3K inhibitor
wortmannin did not affect receptor-induced stimulation of the
CaV1.3 channel at a concentration known to block
class I PI3Ks (data not shown). Also, the involvement of PKA in
transmitting the stimulatory activity from the receptor to the
CaV1.3 channel is unlikely based on independent
and complementary experimental approaches. First, we ascertained that
the human µ-opioid receptor used in the present study couples
exclusively to members of the
Gi/Go family but not to
Gs proteins, which are upstream activators of
adenylyl cyclases and PKA (37). This selective coupling between the
receptor and G proteins was independent of the ligand used. Second, a
stimulation by the downstream effector PKA was ruled out by using the
PKA inhibitor Rp-cAMPS. In contrast, several considerations argue for
an involvement of PKC in stimulation of the
CaV1.3 channel by the
Gi/Go-coupled µ-opioid
receptor. It is well documented that PKC is activated by PT-sensitive
GPCRs via Gi/Go proteins
and phospholipase C (for review see Ref. 52). Accordingly, we confirmed
that the µ-opioid receptor expressed in Xenopus oocytes
was able to activate a calcium-dependent chloride current under
appropriate experimental conditions. This stimulatory effect in
Xenopus oocytes is an established read-out system to detect
coupling between receptors and endogenous phospholipase C, the key
regulator of PKC (53). We also confirmed in a more direct fashion the
ability of PKC to stimulate the CaV1.3 channel
using the PKC activator PMA. Accordingly, recent studies described a
stimulation of L-type calcium channel currents due to phosphorylation
by PKC in the insulin-secreting cell lines HIT-T15 and RINm5F (42, 46).
In conclusion, the present study provides the first detailed
description of a CaV1.3 (L-type
1D) calcium channel subunit cloned from an
insulin-producing cell line. The importance of L-type channels for the
exocytotic process has been stressed by several authors (5, 54). It is
therefore interesting that the neuroendocrine
CaV1.3 and the cardiac
CaV1.2a L-type channel subtypes, which are often
coexpressed, as in insulin-secreting cells (19, 20), display
significant differences with respect to specific biophysical
properties. Differences in the kinetics of channel activation and
inactivation suggest that the two channels may have different roles
during depolarization-induced calcium influx in ß-cells and
consequently in insulin secretion. Most interestingly, this
CaV1.3 isoform is stimulated by GPCRs in a
protein kinase-dependent manner.
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Expression Plasmids and Oocyte Preparation
The identity of the CaV1.3 clone was
verified by sequencing.1
After the addition of an artificial nucleotide sequence that was
constructed to optimize translation, the coding sequence started at
position 9 of the original amino acid sequence (19). No other
differences were found except amino acid positions 427 and 428 of the
original amino acid sequence where we found triplets coding for lysine
and glutamine instead of asparagine and glutamate, respectively. Capped
cRNA transcripts encoding CaV1.3
[XhoI-linearized/T7 RNA polymerase (19)],
CaV1.2a [KpnI/SP6 (10)],
CaV2.2 [SalI/SP6 (13)], and
2-1, termed 2
[SalI/SP6 (10)], ß3
[NotI/T7 (55)] calcium channel subunits as well as the
µ-opioid receptor [Kas I/T7 (56)] were synthesized from the
respective cDNAs using the mMessage mMachine in vitro
transcription kit (Ambion, Inc. Austin, TX). Stage V–VI
oocytes were surgically removed from adult female Xenopus
laevis toads (African Xenopus Facility, Knysa, Republic
of South Africa) anesthetized with 3-aminobenzoic acid ethyl
ester (0.1% wt/vol) according to protocols approved by state and
institutional regulations. The incision was sutured immediately after
removal of the oocytes and the animals returned to the tank after
recovery from surgery. They were then allowed to recover for at least 6
months before being reused as oocyte donors. Follicular cell-free
oocytes were obtained by enzymatic isolation using collagenase type 1A
(2 mg/ml) in Ca2+-free Barth’s solution (in
mM: NaCl, 88; KCl, 1;
NaHCO3, 2.4, MgSO4 , 0.82;
HEPES, 10; pH 7.4 with NaOH) for 2 h and then stored in
Ca2+-containing Barth’s solution (in
mM: NaCl, 88; KCl, 1;
NaHCO3, 2.4; MgSO4, 0.82;
HEPES, 10; CaCl2, 0.41;
Ca(NO3)2, 0.33; pH 7.4 with
NaOH) supplemented with 2.5 mM sodium pyruvate,
0.1% wt/vol BSA, and 100 µg/ml gentamycin at 18 C. The oocytes were
allowed to recover for 24 h and then injected with the same
relative proportions of the appropriate cRNAs at a concentration of 0.1
µg/µl. Using a Nanoject Oocyte Injector (Drummond Scientific Co.,
Broomall, PA), 50 nl of cRNA mixture were injected per oocyte. Barth’s
solution was renewed daily and cells were used on days 2–7 after
injection, depending on the measured current amplitudes and the quality
of the oocytes.
For clarity, the following synonyms were used for the respective
injected subunit combinations: neuroendocrine calcium channel for
CaV1.3/ß3/2,
cardiac CaV1.2a calcium channel for
CaV1.2a/ß3/2,
endogenous oocyte calcium channel for
ß3/2, and neuronal
calcium channel for
CaV2.2/ß3/2.
Electrophysiological Recording
Two-electrode voltage-clamp currents were recorded using an
Oocyte clamp OC-725A amplifier (Warner Instrument Corp., Hamden, CT).
Voltage commands were generated and currents recorded using a personal
computer interfaced with an TL-1 DMA Interface (Axon Instruments,
Foster City, CA) to the amplifier and using the pClamp version 6
software (Axon Instruments). Microelectrodes were filled with 3
M KCl and had typical resistances of 0.5–2.5 M
. The
bath was connected to the clamp circuit via a 3 M KCl-agar
bridge. The bath solution contained in mM:
Ba(OH)2, 35, NaOH, 50; HEPES, 10; pH 7.4 with
methanesulfonic acid. Ba2+ was used as the charge
carrier in all calcium channel experiments. For experiments on
calcium-dependent chloride currents, modified frog Ringer (in
mM: NaCl, 90; KCl, 2.5; CaCl2, 1.8;
HEPES, 10; pH 7.4 with NaOH) was used. Throughout all experiments,
including drug applications, the bath was continuously perfused with
solution at a rate of 2 ml/min. Oocytes were clamped at a holding
potential of -60 mV, in the case of the steady-state inactivation
experiments it was -80 mV. Only those recordings were used where
currents could be adequately voltage clamped. For all experiments
conducted with CaV1.3, CaV1.2a, or
CaV2.2-calcium channels, at least five control oocytes
injected with a corresponding amount of 2
and ß3 cRNA were measured on the same day. Only
those oocyte batches were used for analysis for which the resulting
endogenous calcium current in control oocytes comprised less than 20%
of the total inward barium current in oocytes expressing the exogenous
channels. In the case of experiments where current kinetics were
determined, the maximum endogenous currents were <20% of the total
current. Only those experiments were considered for final analysis that
showed inward holding currents of less than 10% of the maximum inward
current and only minimal fluctuations during the course of the
experiment.
Data Analysis and Presentation
Where appropriate, values are given as means ±
SE. Statistical significance was evaluated by one-way
ANOVA, and individual P values are given. The term n
represents the number of oocytes tested. Curve fitting was done by a
least-squares minimization method using the following equations. For
I-V curves:
 | (1) |
 | (2) |
 | (3) |
 | (5) |
is the time (t) at which the current had
decreased to 1/e of its initial amplitude, A is the maximal amplitude
of the component, and C is the steady-state current levels.
Dose-response curves for (+)isradipine were fitted applying
 | (6) |
DAMGO was used to activate the µ-opioid receptor expressed after coinjection of its cRNA. The resulting effect on each current was calculated between the last data point before agonist application and the point of maximal effect. Current run-down was not taken into account.
Note Added in Proof
While this paper was in press, Striessnig and co-workers (57)
published biophysical and pharmacological properties of an
1D subunit from cochlea inner hair cells.
|
ACKNOWLEDGMENTS |
|---|
2; Dr. Y.
Fujita (Matsushita Electric Industrial Co., Seika, Japan),
CaV2.2; and Dr. L. Emorine (Institut de
Pharmacologie et de Biologie Structurale, Toulouse, France), µ-opioid
receptor. We thank Dr. Günther Schultz for continuous support. We
also thank Drs. A. Zobel, C. Canti, and A. G. Obukhov for
introduction into the Xenopus laevis oocyte model, as well
as A. Tomschegg for technical assistance. |
FOOTNOTES |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft, Royal Society, and Fonds der Chemischen Industrie.
1 The nucleotide sequence data have been submitted
to the EMBL Nucleotide Sequence Database under accession number
AJ311617. 
Received for publication June 29, 2000. Revision received March 12, 2001. Accepted for publication April 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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; n =
24), -24.7 mV for the cardiac CaV1.2a (
; n = 20),
and -38.5 mV for the endogenous (
; n = 7) calcium channel.
) and 43 nM
for the cardiac CaV1.2a (
) calcium channel. The
pulse frequency was 0.2 Hz. B, Maximum current changes under DAMGO
application (2.5 min) for the neuronal (n = 7), the endogenous
(n = 23), and the neuroendocrine CaV1.3 (n = 26)
calcium channel. The fourth column shows the DAMGO effect on the
neuroendocrine CaV1.3 channel after preincubation with H7
(100 µM; n = 5); the fifth column shows the DAMGO
effect on this channel after preincubation with bisindolylmaleimide I
(Bis I; 100 nM; n = 5). Both inhibitors were also
present during the DAMGO application in the respective experiments.