Functional Role of Alternative Splicing in Pituitary P2X2 Receptor-Channel Activation and Desensitization
Taka-aki Koshimizu1,
Melanija Tomi
,
Fredrick Van Goor and
Stanko S. Stojilkovic
Endocrinology and Reproduction Research Branch National
Institute of Child Health and Human Development National Intitutes
of Health Bethesda, Maryland 20892
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ABSTRACT
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Although ATP-gated ion channel (P2XR) expression
is high among anterior pituitary cells, identification of the receptor
subtypes and their selective expression within subpopulations of cell
types, as well as their physiological role(s), are incompletely
characterized. In this study, we focused on the expression and activity
of the P2X2R subtype in anterior pituitary
cells. Our results indicate that the primary
P2X2R gene transcript in pituitary cells
undergoes extensive alternative splicing, with generation of six
isoforms. Two of these isoforms encode functional channels when
expressed in GT1 or HEK293 cells: the wild-type
P2X2R and the spliced isoform
P2X2-2R, which lacks a stretch of
carboxyl-terminal amino acids
(Val370-Gln438). Four
other clones showed different alterations, including an interfered
reading frame starting in the first transmembrane domain and a 27-amino
acid deletion in the large extracellular loop. When expressed
separately or in combination with wild-type channels, these clones were
nonfunctional. In single cell Ca2+ current and
cytosolic Ca2+ concentration
([Ca2+]i)
measurements, the P2X2R and
P2X2-2R had similar
EC50 values for ATP and time courses for
activation and recovery from desensitization but differed significantly
in their desensitization rates. The spliced isoform exhibited rapid and
complete desensitization, whereas the wild-type channel desensitized
slowly and incompletely. The mRNAs for wild-type and spliced channels
were identified in enriched somatotroph, but not gonadotroph or
lactotroph fractions. Expression of a functional ATP-gated channel in
somatotrophs was confirmed by the ability of ATP to increase the
frequency of [Ca2+]i
spikes in spontaneously active cells or initiate spiking in quiescent
cells. When voltage-gated Ca2+ influx was
blocked, ATP increased
[Ca2+]i, with a
similar profile and EC50 to those observed in
GT1 cells heterologously expressing wild-type or spliced
P2X2R. The ligand-selectivity profile of native
channels was consistent with the presence of
P2X2R in somatotrophs. Finally, the
desensitization rate of P2X2R in a majority of
somatotrophs was comparable to that observed in neurons coexpressing
wild-type and spliced channels. These data indicate that alternative
splicing of P2X2R and coexpression of
P2X2R and P2X2-2R
subunits provide effective mechanisms for controlled cationic influx in
somatotrophs.
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INTRODUCTION
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Extracellular ATP acts on metabotropic P2 receptors (P2YR) and
ionotropic P2 receptor channels (P2XR), which represent two
structurally and functionally distinct purinergic receptor types (1).
P2YR are members of the heptahelical G protein-coupled family of
Ca2+-mobilizing receptors (2). P2XR belong to a new class
of ligand-gated channels. These channels have two putative
transmembrane domains (M1 and M2) with an intervening hydrophilic loop
of about 300 amino acids and an adjacent hydrophobic segment (H5) and
cytoplasmic amino and carboxyl termini (3). Both P2YR and P2XR are
expressed in neuronal and nonneuronal tissues, including rat pituitary
cells. The structure and signal transduction pathways of pituitary P2YR
are well characterized. Molecular studies identified that the
P2Y2 subtype of these receptors is expressed in pituitary
tissue (4). Their activation leads to an increase in inositol phosphate
turnover (5) associated with an increase in cytosolic calcium
concentration ([Ca2+]i) (6, 7) and
translocation of protein kinase C (8).
In contrast, our understanding of the P2X-signaling pathway in the
pituitary is incomplete. Two recently published reports (9, 10)
indicate the expression of an operative P2XR in pituitary cells. An
intense signal for P2X2R on RNA blot was seen from
pituitary, and in situ hybridization analysis suggested that
specific signals were present in both intermediate and anterior lobes
(9). Pharmacological studies further indicate the expression of a
Ca2+-conducting P2X2R and/or P2X5R
in pituitary gonadotrophs and other unidentified pituitary cell types.
These channels can remodulate spontaneous electrical activity and
associated Ca2+ entry through voltage-gated calcium
channels, but also conduct Ca2+ in cells when voltage-gated
Ca2+ influx is inhibited (10). In this study, we
characterized the cell type-selective expression of the
P2X2R within the anterior pituitary and their potential
physiological role in cellular Ca2+ homeostasis.
Furthermore, we compared the activation-desensitization kinetics and
pharmacological profiles of native and cloned P2X2R.
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RESULTS
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Molecular Cloning and Sequence Analysis of Pituitary
P2X2R cDNAs
The possible expression of P2X5R and P2X2R
in rat pituitary cells was determined at the transcriptional level
using RT-PCR. Oligonucleotide primers were designed in 3'- and
5'-untranslated regions, and transcripts carrying the entire coding
region of the channel proteins were amplified. In mixed populations of
pituitary cells, only a low signal for P2X5R was detected
(data not shown). In contrast, abundant PCR products of two different
sizes, approximately 1.6 and 1.4 kb long (Fig. 1A
, lane 3), were consistently amplified
by the P2X2R primers. The intensity of these two bands
remained equal on the gel after the annealing temperature in the PCR
was increased to 60 C. Higher annealing temperatures resulted in no
detectable PCR products. This observation suggested that at least two
distinct lengths of P2X2R transcripts, with the same
primer-annealing profiles, are expressed in rat pituitary cells.

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Figure 1. Expression of P2X2R Variants in the
Anterior Pituitary
RT-PCR analysis of total RNA from primary cultures of mixed pituitary
cells and transient transfection of GT1 neurons were performed as
described (Materials and Methods). Oligonucleotide
primers specific for the P2X2R sequence used are U1 and L1
for experiments shown in panel A and U530 and LD5 for experiments shown
in panel B. The 5-µl aliquots of the PCR reaction were analyzed on a
1% (panel A) and 2% (panel B) agarose gel containing ethidium
bromide. The plasmid (500 pg) containing each cDNA was added for a
positive control in PCR. GAPDH, Glyceraldehyde phosphate
dehydrogenase.
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To test whether the alternative splicing of the primary
P2X2R gene transcript accounts for the occurrence of the
two bands, the PCR products were purified separately from the gel and
subcloned into a pBluescript vector. Restriction endonuclease mapping
and nucleotide sequence analysis of the subcloned fragments (total 21
clones) showed two major inserts, the P2X2R (9) in the
upper band and the P2X2-2R (11, 12) in the lower band. In
addition, four minor clones, termed here clone 3, 4, 5, and 6, were
isolated (Fig. 2
).
(The assigned accession numbers by GenBank for clones 3 to 6 are
AF020756, AF020757, AF020758, and AF020759, respectively.)
All splicing donor and acceptor sites are consistent with the GT/AG
rule (13). The 81-bp deletion observed in clones 3, 4, and 5 was
considered to have occurred together with adjacent introns (Table 1
). The insertion sequences in clone 4
(46 bp) and clone 5 (140 bp) resemble the genomic sequence (intron I)
reported by Brandle et al. (11). We also observed three
nucleotide additions in the corresponding sequence of clone 4
(C171, G182, and C183) and clone 5
(C265, G276, and C277). The deduced
amino acid sequences indicated that the P2X2-2R lacks a
69-amino acid segment from the intracellular C terminus, whereas clone
3 lacks 27 amino acids of the extracellular part (Fig. 3
). Nucleotide sequences of clones 4, 5,
and 6 were changed within those corresponding to the first
transmembrane domain of P2X2R, resulting in frame shifts
and premature stop codons (Fig. 3
).

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Figure 3. Alignment of the Deduced Amino Acid Sequences for
the P2X2R and Spliced Isoforms
The amino acid sequences of spliced forms identical to those of
P2X2R are shaded. The predicted
two-transmembrane segments (M1 and M2) and a segment resembling the H5
region of voltage-gated potassium channels are indicated by
solid lines.
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Expression of P2X2R cDNAs in Mammalian
Cells
The pME18Sf- mammalian expression vector carrying
cDNA for each transcript was transiently transfected in GT1 neurons,
and the expression of functional Ca2+-conducting channels
was evaluated by single-cell [Ca2+]i
measurements. Application of 100 µM ATP was associated
with a rapid and sustained increase in
[Ca2+]i in cells expressing wild-type
P2X2R (termed here GT1/P2X2R cells) and spliced
P2X2-2R (termed here GT1/P2X2-2R cells) (Fig. 4A
, two bottom tracings). In
contrast, cells transfected with one of the minor clones (3 to 6) did
not express functional Ca2+-conducting channels, as no
increase in [Ca2+]i was observed after
addition of 100 µM ATP (Fig. 4A
, four top
tracings) or 500 µM ATP (data not shown). We also
tested the hypothesis that clones 3 to 6 may form functional
heteropolymers with P2X2R. For this purpose, GT1 neurons
were cotransfected with cDNA for one of these clones, or an empty
vector together with the P2X2R cDNA. In all cases, the
percentage of cells expressing functional channels was the same
(3040%). Furthermore, difference in the pattern of ATP-induced
Ca2+ signaling in responsive cells was indistinguishable
from those transfected with P2X2R cDNA plus empty
vector.

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Figure 4. Patterns of Ca2+ Entry through Rat
Pituitary P2X2R and Its Isoforms
A, Representative traces of the ATP-induced Ca2+ signal in
GT1 neurons expressing one of the six clones (see Figs. 2 and 3 ). B and
C, Comparison of the activation (B) and desensitization (C) patterns of
the ATP-induced signal between the wild-type (P2X2R) and
functional splice variant (P2X2-2R). Representative
ICa (left panels) and
[Ca2+]i (right panels) traces
are shown. HEK293 and GT1 cells were used for the measurement of
ICa and [Ca2+]i, respectively.
Arrow represents time of ATP application.
Bar indicates duration of ATP application.
ICa measurements were done in cells clamped at -70 mV.
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Subunit specific expression in GT1/P2X2R and
GT1/P2X2-2R cells was confirmed by RT-PCR. Figure 1A
shows
different sizes of PCR products amplified from cells transfected with
either wild-type or spliced variant cDNAs. The low molecular weight PCR
fragment corresponding to the deleted P2X2-2R transcript
was not detected in GT1/P2X2R cells (lane 1) and vice versa
(lane 2). Also, a 81-bp deletion in clone 3, but not in
P2X2R or P2X2-2R, was detected only from
native pituitary and GT1/clone 3 cells (Fig. 1B
). Thus, our
heterologous expression system allows the examination of channel
subunit-specific characters.
Activation, Desensitization, and Recovery of
P2X2R and P2X2-2R
To study the gating properties of the P2X2R and
P2X2-2R, both Ca2+ current (ICa)
and [Ca2+]i measurements were employed using
HEK293 cells and GT1 neurons expressing these channels. Figure 4
, B and
C, illustrate the kinetics of activation (increasing phase) and
desensitization (decreasing phase) of these channels in HEK293
(left panels) and GT1 neurons (right panels)
stimulated with 100 µM ATP. Both ICa and
[Ca2+]i measurements indicate that channel
activation is rapid (Fig. 4B
). In contrast to the activation rates,
there was a consistent difference in the pattern of desensitization
between the two channels. The P2X2-2R desensitized
completely within a few minutes after addition of 100 µM
ATP, whereas the P2X2R exhibited slow-desensitizing
signals. This was consistently observed in both ICa (Fig. 4C
, left panel) and [Ca2+]i
measurements (Fig. 4C
, right panel).
In further experiments on the activation and desensitization properties
of these channels, single-cell [Ca2+]i
measurements were employed. For both GT1/P2X2R and
GT1/P2X2-2R cells, amplitude-modulated changes in
[Ca2+]i were observed in response to
increasing concentrations of ATP from 10 µM to 500
µM (Fig. 5
). The calculated
EC50 values were about 10 µM and 13
µM, and the Hill coefficients were 1.7 and 2.2 for
P2X2R and P2X2-2R, respectively. In addition,
there was not a significant difference in the peak responses to ATP
between the two channels (Fig. 5B
). Figure 5A
also illustrates that the
activation and desensitization rates decreased progressively with an
increase in ATP concentration. At a supramaximal (500 µM)
concentration of ATP, the wild-type channel remained slowly
desensitizing, indicating that the rapid desensitization of the spliced
isoform is not a consequence of its sensitivity to ATP, but represents
an intrinsic characteristic of this channel.

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Figure 5. Concentration Dependence of ATP on
[Ca2+]i in GT1 Neurons Expressing
P2X2R and P2X2-2R
A, Representative tracings at each ATP concentration from a single
experiment. B, Averaged data (mean ± SEM; n
10) expressed as percent basal [maximum
F340/F380 response subtracted by a basal value
of 0.42 ± 0.05]. Results were fitted to a four-parameter
logistic equation, from which the EC50 values and Hill
coefficients (nH) were derived.
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To quantitatively describe activation and desensitization kinetics of
the wild-type and spliced channels, the rapid increasing activation
phase and the subsequent desensitization phase of
[Ca2+]i responses during continuous
stimulation with ATP were fitted separately with the sums of
exponential functions. At lower concentrations of ATP, one exponential
component was sufficient to describe activation of wild-type and
spliced channels (data not shown). At medium to higher ATP
concentrations, the exponential fittings were not satisfactory, because
of the low number of experimental points in the rapid activation phase
limited by the resolution time in two-wavelength
[Ca2+]i measurements (one point per second).
As a result, we were unable to analyze the relationship between the
activation rate and ATP concentration for the two channels. Instead, we
compared the mean times needed to reach the peak response (activation
time) during stimulation of these two channels with increasing
concentrations of ATP (Fig. 6A
). The
half-maximal activation times were at about 50 µM,
suggesting that ATP binds with low affinity to both channels. One
exponential component was also sufficient to fit the subsequent decline
in [Ca2+]i (desensitization) in the continued
presence of ATP. Figure 6B
shows representative
[Ca2+]i tracings and rate constants of
desensitization for P2X2R and P2X2-2R in
response to 100 µM ATP. There was a consistent 10-fold
difference in the rates of desensitization for these two channels. The
mean half-times of desensitization of P2X2R and
P2X2-2R were 187 ± 40 sec (n = 10) and 18
± 3 sec (n = 17), P < 0.001.
Recovery from ATP-induced desensitization of the wild-type and spliced
channels was investigated in cells exposed to two 5-min pulses of 100
µM ATP, with the interpulse interval varied between 2 and
15 min (Fig. 7A
). The calculated
half-times for recovery of these two channels were comparable (Fig. 7B
). A similar time-dependent recovery from ATP-induced activation of
ICa was observed in HEK293 cells expressing
P2X2R (data not shown). These observations are consistent
with the conclusion that the missing 69-amino acid segment in the
C-terminal of P2X2-2R is not essential for activation but
affects the rate of desensitization of these channels. Furthermore, the
results indicate that the rapid desensitization of P2X2-2R
does not shut off these channels for a prolonged time. Thus, the
splicing of P2X2R provides an effective mechanism for the
controlled Ca2+ influx without affecting the activation and
recovery profiles.

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Figure 7. Recovery from ATP-Induced Desensitization of
P2X2R and P2X2-2R Expressed in GT1 Neurons
A, Representative tracings of [Ca2+]i from
cells exposed to two 5-min pulses of 100 µM ATP. The
interpulse interval ( t) varied between 2 and 15 min.
Each trace represents the mean ratio
[F340/F380] from at least five separate
cells. Basal levels before ATP addition are indicated by the
dotted lines. The bath was continuously perifused at a
rate of 2 ml/min using a gravity-driven superfusion system, the outflow
of which was placed near the cells resulting in complete solution
exchange around the cells within 10 sec. B, Averaged data (means
± SEM; n 5). The results were expressed as
percentage control, using the peak [Ca2+]i
values during the two ATP pulses. The half-times were derived from
fitted logistic curves.
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Selective Expression of P2X2R in Pituitary
Cells
To address the distribution of P2X2R among
subpopulations of pituitary cell types, RT-PCR analysis and
[Ca2+]i measurements were performed using
enriched populations of specific hormone-secreting cell types. The
Ficoll gradient procedure for purification leads to 15 fractions of
anterior pituitary cells. As shown in Fig. 8A
, the first four fractions are enriched
with gonadotrophs, fractions 59 with somatotrophs, and fractions
1115 with lactotrophs. The transcripts for P2X2R were
observed in somatotroph, but not in gonadotroph and lactotroph,
fractions of anterior pituitary cells (Fig. 8B
).

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Figure 8. Distribution of P2X2R in Anterior
Pituitary Cell Subpopulations
A, Purification of gonadotrophs, somatotrophs, and lactotrophs from
mixed anterior pituitary cells. Dispersed cells from ovariectomized
rats were separated by Ficoll gradient into 15 fractions. The LH, GH,
and PRL contents were determined in all fractions. The values shown
represent the hormone content determined in 100,000 cells multiplied by
the number of cells per fraction. The shaded areas
indicate cell fractions used for RT-PCR analysis. B, The same volume of
cDNA samples from purified gonadotrophs, lactotrophs, somatotrophs, and
mixed populations of anterior pituitary cells was used for PCR
reactions employing the P2X2R-specific primer U1 and L1.
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Somatotrophs exhibit spontaneous and extracellular calcium-dependent
[Ca2+]i transients (Fig. 9
), with a spiking amplitude 3- to 5-fold
higher than that of gonadotrophs and lactotrophs (14). In addition to
the high amplitude [Ca2+]i spikes, these
cells were further identified by their response to somatostatin and by
the lack of responses to TRH (typical for lactotrophs and thyrotrophs),
GnRH (typical for gonadotrophs), and vasopressin (typical for
corticotrophs). In somatotrophs, 100 µM ATP increased the
frequency, but not the amplitude, of [Ca2+]i
spiking (Fig. 9A
). Stimulatory effects of ATP on the frequency of
[Ca2+]i transients were consistently observed
in 5100 µM concentration range. At higher
concentrations of ATP, nonoscillatory elevations in
[Ca2+]i were also observed.

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Figure 9. Characterization of ATP-Induced
[Ca2+]i Responses in Single Somatotrophs
A, Representative tracings from three separate cells of the ATP-induced
increase in frequency of spontaneous [Ca2+]i
transients. ST, Somatostatin. B, Extracellular Ca2+
dependence and ligand-selectivity profile of P2X channels expressed in
somatotrophs. Upper tracings, Cells were bathed in
Ca2+-containing (+Ca2+) or
Ca2+-deficient (-Ca2+) medium. To abolish
spontaneous [Ca2+]i transients, all
recordings were done in the presence of 50 µM
Cd2+.
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Removal of extracellular Ca2+ abolished spontaneous
[Ca2+]i transients. In such cells, ATP was
unable to increase [Ca2+]i (Fig. 9B
, upper panel, -Ca2+), confirming that
somatotrophs express P2XR, but not P2Y, calcium-mobilizing receptors.
Thus, Ca2+ influx through ATP-gated channels is exclusively
responsible for the ATP-induced modulation of spike frequency. In
further experiments, spontaneous electrical activity and associated
[Ca2+]i transients were abolished by addition
of 50 µM Cd2+, a blocker of voltage-gated
calcium channels. The subsequent addition of ATP in these cells led to
a nonoscillatory [Ca2+]i response (Fig. 9B
, upper panel, +Ca2+). ATP, ATP
S, and 2
methylthio-ATP were found to be equipotent as agonists, whereas BzATP
and ADP were less potent (Fig. 9B
). Conversely, adenosine, AMP,
,ß-methylene-ATP, ß,
-methylene ATP, and UTP were unable to
alter [Ca2+]i in somatotrophs. Similar
ligand-selectivity profiles were observed in GT1/P2X2R and
GT1/P2X2-2R cells. Thus, somatotrophs express ATP-gated
channels comparable to P2X2R, which can alter the frequency
of spontaneous [Ca2+]i transients when
activated at physiological ATP concentration.
Native vs. Cloned Channels
Desensitization properties of P2X2R expressed in
somatotrophs were studied in cells with inhibited voltage-gated
Ca2+ influx by 50 µM Cd2+. As in
GT1 neurons expressing wild-type and spliced channels, single
exponential fittings were sufficient to describe the desensitization
characteristics of native channels in somatotrophs. However, the rates
of desensitization in somatotrophs differed significantly from that
observed in cells expressing exclusively P2X2R or
P2X2-2R. As shown in Fig. 6
, the ATP-induced
[Ca2+]i responses in GT1/P2X2R
and GT1/P2X2-2R cells were highly homogeneous, with a
consistent 10-fold difference in the desensitization rates [(k
(s-1): 0.0037 ± 0.0008 (n = 10) for
P2X2R vs. 0.0381 ± 0.0064 (n = 17)
for P2X2-2R]. Conversely, the desensitization rates in
somatotrophs varied from 0.0051 to 0.0585 s-1 (Fig. 10
, left panels). Thus, the
rates of desensitization were in the range between those observed in
GT1/P2X2R and GT1/P2X2-2R cells.

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Figure 10. Comparison of the Patterns of Desensitization
Evoked by 100 µM ATP in Somatotrophs and GT1 Neurons
Cotransfected with P2X2R and P2X2-2R
A, Examples of ATP-induced [Ca2+]i profiles
in somatotrophs in the presence of 50 µM Cd2+
(which was added to inhibit votage-gated Ca2+ influx). B,
GT1 neurons were transfected with a constant total amount of plasmid
DNA, but with variable ratios for P2X2R and
P2X2-2R, as indicated above each tracing.
Representative tracings observed for each ratio of
P2X2/P2X2-2 are shown (dotted
lines). Each tracing was fitted with a single exponential
(solid line), which is extended in upper direction for
clarity. The values above the curves indicate the
calculated rates of desensitization.
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Since the transcripts for P2X2R and P2X2-2R
were equally abundant (Fig. 1
) and consistently present in all
pituitary preparations, we speculate that the coexpression of wild-type
and spliced channels in a single cell leads to such a pattern of
signaling. To test this hypothesis, GT1 neurons were transfected with
cDNAs for both P2X2R and P2X2-2R in
different ratios, keeping the total amount of the expression plasmids
unchanged. In such cells, the pattern of ATP-evoked Ca2+
signals was between those occurring in the wild-type and spliced
channels, and comparable to that observed in pituitary somatotrophs.
Figure 10B
illustrates the most frequent profiles of
[Ca2+]i responses in cells that were
transfected with cDNAs for both channels. There is a progressive shift
in the half-times of desensitization with an increase in the amount of
transfected P2X2-2R plasmids. In all cases, a single
exponential function satisfactorily described the ATP-induced
[Ca2+]i profiles. This suggests that
heteromultimers, rather than coexpression of homomultimers, occurs in
cells expressing both isoforms. The similarity in the profiles of
[Ca2+]i transients in somatotrophs and
transfected GT1 neurons further indicates that native channels are also
heteromultimers composed of wild-type and spliced subunits.
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DISCUSSION
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Three classes of ligand-gated ionic channels, defined by their
molecular architecture, are expressed in mammalian cells. The first
class is represented by the four-transmembrane domain family of
receptor channels that are specific for cations (nicotinic and
5-hydroxytryptamine) or anions (
-aminobutyric acid and glycine
channels). The second contains glutamate receptor channels and is
composed of
-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic
acid (AMPA), kainate, and N-methyl-D-aspartate
(NMDA) subfamilies. These receptor channels share three hydrophobic
segments and are expressed exclusively in the central nervous system
(15). The third class of receptor channels is gated by ATP. Seven
subunits of P2XR have been recently cloned and named P2X1
to P2X7 (9, 16, 17, 18, 19, 20, 21). The overall topology suggest that each
channel subunit has only two transmembrane domains and resembles the
inward rectifier potassium channels, the amiloride-sensitive
sodium channels, and the mechano-sensitive ionic channels. There is
3550% identity and 5065% similarity between pairs of P2XR. The
expressed receptors differ among themselves with respect to the action
of ATP analogs, desensitization rates, and antagonist effectiveness (3, 22).
The P2X1R and P2X3R cation-selective channels
show relatively high calcium vs. sodium permeability
(PCa/PNa = 4). The additional common
characteristics of these two channels are their sensitivity to
,ß-methylene-ATP and to the P2XR antagonist, suramin, as well as
their rapid desensitization to high agonist concentrations (16, 17, 18).
Other members of these ligand-gated receptor channels do not respond to
,ß-methylene-ATP and desensitize slowly compared with
P2X1R and P2X3R. Three of them,
P2X4R, P2X6R, and P2X7R, are not
sensitive to suramin, whereas P2X2R and P2X5R
are (19, 20, 21). The P2X2R subtype has a relatively long C
terminus composed of 119 amino acids. The pharmacological profile of
P2X2R resembles that of native P2XR on PC12 cells and some
neurons (9). Northern blot analysis suggests expression of
P2X2R in the brain, spinal cord, intestine, and vas
deferens. The most intense signal was observed in the pituitary (9);
however, only limited information concerning the functional role of
these channels in pituitary cells was available.
Our cloning study indicates that multiple transcripts for
P2X2R are present in anterior pituitary cells. Two of them
are the P2X2R and P2X2-2R, the structures of
which have been reported in other tissues (11, 12). Clones 36,
described here, are novel, and several additional isoforms were
reported by others (12, 23), suggesting that the presence of multiple
transcripts is not unique to pituitary cells. In general, such
transcripts are formed by the posttranscriptional processing of the
primary P2X2R gene transcripts (24). Three lines of
evidence indicate that P2X2R isoforms observed in the
pituitary represent the transcripts after the actual mRNA processing.
In the first strand cDNA synthesis, the transcripts having a poly (A)
tail were reverse-transcribed with oligo d(T)12-18
priming. Also, our set of the P2X2-specific primers
amplifies the entire coding region for the original P2X2R
protein during a single PCR reaction. Finally, when compared with the
P2X2R gene structure, a part of the exonic sequence for
P2X2R was deleted in each P2X2R isoform, and
all of the splicing donor and acceptor sites are consistent with the
GT-AG rule (13). The structure of clones 4, 5, and 6 clearly indicate
that alternative splicing of primary mRNA is responsible for their
generation. Although the P2X2-2R and clone 3 could also be
produced in native cells by a splicing reaction of the wild-type mRNAs,
this is unlikely to occur in GT1 neurons since GT1/P2X2R
cells only produced the corresponding P2X2R transcript.
This suggests that the same potential splicing sites are recognized in
a different way by native pituitary and GT1 cells.
Both the P2X2R and P2X2-2R spliced forms
encode functional channels when expressed in GT1 neurons or HEK293
cells. When clones 36 were expressed in GT1 neurons, they did not
form a Ca2+-conducting channel. Clones 4, 5, and 6 encode
proteins that are missing the H5 and M2 domains, as well as the C
terminus, so their inability to conduct Ca2+ was not
surprising. Clone 3 is missing a stretch of the extracellular domain
that encodes residues Asn173-Ser199, a part of
the potential ATP-binding motif (9), which may explain the lack of
expression of a functional channel by this transcript. It is also
unlikely that one of the proteins derived from these clones together
with P2X2R proteins assembles a functional heteromultimer
channel with altered binding characteristics and/or conductivity. On
the other hand, the production of these transcripts by alternative
splicing reduces the relative amount of the final mRNA for functional
P2X2R channel molecules in cells. This is consistent with
the hypothesis that alternative splicing also provides a mechanism for
regulating P2X2R gene expression in pituitary cells.
Identification of the P2XR-signaling pathway(s) and their role(s)
in the control of Ca2+ homeostasis in pituitary cells have
not been fully characterized. To date, there is a lack of information
on the subtypes of P2XR expressed in the anterior pituitary and their
selective expression among the subpopulations of anterior pituitary
cells. We have recently reported that the
P2X2/P2X5-like receptor channel is expressed in
pituitary gonadotrophs and several other unidentified pituitary cell
types (10). Our PCR analysis, however, argues against the hypothesis
that the P2X2R subtype is expressed in these cells. It is
also unlikely that lactotroph subpopulations express these channels.
The signal was specifically associated with somatotroph-enriched
fractions of pituitary cells. Since the somatotrophs are the most
abundant subpopulation of anterior pituitary cells, this provides a
rationale for the observation by Brake et al. (9) concerning
the presence of intense signals in anterior pituitary tissue.
Single-cell [Ca2+]i measurements also
support the existence of ATP-gated channels in somatotrophs, activation
of which leads to an increase in [Ca2+]i
spike frequency. In addition, the ATP-induced rise in
[Ca2+]i was observed in somatotrophs exposed
to the inorganic voltage-gated calcium channel blocker,
Cd2+. This observation indicates that native channels are
capable of conducting Ca2+ independently of voltage-gated
calcium channels, which are spontaneously operative in these cells. The
cationic influx through native P2X channels in somatotrophs has the
capacity to remodulate spontaneous electrical activity and associated
voltage-gated Ca2+ influx. We may then speculate that
voltage- and ATP-gated channels act in a coordinated manner controlling
both Ca2+ signaling and Ca2+-dependent cellular
functions in these cells.
A comparative study on behavior of the native channels and
expressed wild-type and spliced channels further indicates that they do
not differ in terms of their ligand selectivity profiles, activation
properties, EC50 values, maximum
[Ca2+]i responses to ATP, or the recovery
(from desensitization) times. However, the native channels exhibited
patterns of desensitization that differed from those observed in
wild-type and spliced channels. The wild-type channels desensitized
slowly and incompletely, whereas the spliced channels desensitized
rapidly and completely. This is in accord with recently published
observations in other tissues (11, 12). From a physiological point of
view, the rapidly desensitizing current through P2X2-2R
represents an advantage rather than a disadvantage. Activation of
voltage-gated channels, including Ca2+ channels, is
commonly associated with inactivation that terminates or attenuates the
currents (15), and such inactivation is critically important to protect
the cells from overloading with Ca2+. In this regard,
desensitization of P2X2-2R is a process analogous to the
inactivation of voltage-gated channels.
Recently, two experimental approaches have been used to
characterize the molecular mechanism of P2XR desensitization: the
construction of chimeric channels between slowly (P2X2) and
rapidly (P2X1 and P2X3) desensitizing subtypes
and coexpression of different subtypes of these channels. Chimeric
studies suggest that the responsible domains for desensitization are
localized within the two transmembrane regions of P2X1R and
P2X3R (25). The coexpression of P2X2R and
P2X3R was also found to account for the desensitization
properties of P2XR channels expressed in sensory neurons (17). Our
results with coexpression of wild-type and spliced channels are also
consistent with the assembly of functional heteropolymers. Furthermore,
such channels show an enhanced rate of desensitization that is
physiologically relevant for controlled Ca2+ influx. Our
results also suggest that native channels expressed in somatotrophs are
heteropolymers. Thus, the coexpression of wild-type and spliced
channels provides an effective mechanism by which to sustain
Ca2+ signaling and protect cells from overloading with
Ca2+.
In conclusion, our results demonstrate, for the first time, that
somatotrophs express the P2X2R subtype of ATP-gated
channels, activation of which leads to modulation of action
potential-dependent calcium transients. Molecular cloning and
expression of the P2X2R revealed two physiologically
important characteristics: a relatively high Ca2+
permeability and a slowly desensitizing current. Because of their slow
desensitization, prolonged activation of P2X2R would lead
to a sustained increase in [Ca2+]i that is
potentially harmful for the cells. However, somatotrophs exhibit a
controlled Ca2+ influx during the sustained agonist
stimulation. This is achieved by alternative splicing of
P2X2R and coexpression of wild-type and spliced channels.
When expressed separately, the spliced P2X2-2R subunits
form a functional channel, which rapidly desensitized. When
co-expressed with the wild-type, it effectively decreased
Ca2+ influx in single cells during prolonged agonist
stimulation in a manner highly comparable with that observed in native
channels.
 |
MATERIALS AND METHODS
|
---|
Tissues and Cell Cultures
GT1 immortalized neurons were employed for
[Ca2+]i measurements since they showed no
functional response to ATP. HEK293 cells bathed in
Ca2+-deficient medium responded to 100 µM ATP
by an increase in [Ca2+]i, indicating the
expression of a native P2Y receptor, and were employed only for
ICa measurements. GT1 neurons were cultured in DMEM and
Hams F-12 medium (1:1) supplemented with 10% FCS, and HEK293 cells
were cultured in MEM supplemented with 10% FCS (GIBCO, Rockville, MD).
Single-cell [Ca2+]i measurements were also
performed on anterior pituitary cells from female Sprague Dawley rats
obtained from Charles River Inc. (Wilmington, MA). Dispersed pituitary
cells were cultured in ATP-deficient medium 199 containing Earles
salts, sodium bicarbonate, 10% horse serum, and antibiotics. Cell
purification of dispersed pituitary cells from ovariectomized rats was
done by sedimentation on a Ficoll gradient as previously described
(26).
RT-PCR Analysis of P2X2R
Total RNA was isolated from pituitary primary cell cultures and
GT1 cells using TRIZOL reagent (GIBCO). First-strand cDNA was
synthesized from 5 µg total RNA, using Superscript II reverse
transcriptase and oligo(dT)12-18 primers (GIBCO) in a
reaction volume of 20 µl. A 1-µl aliquot of the resulting
single-strand cDNA was used in the PCRs, which were performed in 12.5
µl, containing 200 µM each of four deoxynucleotide
triphosphates, 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 2 mM MgCl2, and 0.25 U of Ex
Taq polymerase (PanVera Corporation, Madison, WI). The PCR
primers covering the entire protein-coding sequence for
P2X2R (9), as well as its consensus sequence for
translation initiation (24), had the following sequences: sense primer
U1, 5'-TTCCCGCGGGGGCGGCCAT-3'; antisense primer L1,
5'-TCGTTTCTGTTTCCCAGTCACAT-3', as indicated by arrows in
Fig. 2
. For detection of the 81-bp deletion in clones 3, 4, and 5,
sense primer L530, 5'-CGGTGGAGGATGGAACTTCTGAC-3', and
antisense primer LD20, 5'-CACCTTGTCGAACTTCTTATGG-3', were
used. The P2X5R-specific primers were:
P2X5RU1, 5'-CGCCAGAGTGAGCTGGAGGC-3'; and
P2X5RL1, 5'-GGAGTTGAGGGGCTTTTCTC-3'. Reactions were run for
30 cycles at 94 C, 1 min (denaturation); 57 C, 35 sec (annealing); 72
C, 1.5 min (extension); followed by a final extension for 10 min at 72
C. Amplified DNA fragments were electrophoresed on agarose gel and
visualized with ethidium bromide. The same volume of samples used for
P2X2R mRNA analysis were also subjected to PCR using
glyceraldehyde phosphate dehydrogenase-specific primers (27); sequences
for sense and antisense primers were 5'-GGCATCCTGGGCTACACTG-3' and
5'-TGAGGTCCACCACCCTGTT-3', respectively. No PCR products were
detected from controls containing all components except reverse
transcriptase, ruling out the possibility of genomic DNA
contamination.
Isolation of cDNA Encoding P2X2R
Transcripts
The PCR products amplified with P2X2R primers showed
two bands on the agarose gel: the expected 1.6-kb fragment and a
smaller 1.4-kbp DNA fragment (Fig. 1
, lane 3). These two DNA fragments
were recovered separately and inserted into the pBluescript SK(-)
plasmid (Stratagene, La Jolla, CA). The sequence of each clone was
determined by the dideoxy chain termination method, using Sequenase
version 2.0 (Amersham, Arlington Heights, IL). For each transcript, at
least two independent clones derived from separate PCR reactions were
sequenced on both strands.
Expression of the P2X2R in GT1 and HEK296
Cells
The subcloned cDNA inserts were digested with XhoI
and NotI (New England Biolabs, Inc., Beverly, MA) and
ligated into XhoI and NotI sites of the
expression vector pME18Sf-. On the day of transfection, 3
µg of the plasmid DNA were mixed with 7 µl Lipofectamine in 3 ml
serum-free OPTI-MEM medium (GIBCO), incubated for 20 min at room
temperature, and then applied to cells (106 cells per 60-mm
dish). After 6 h incubation, the medium was replaced with fresh
culture medium and the cells were allowed to grow for 24 h. For
single-cell calcium recordings, transfected cells were plated on
poly-l-lysine-treated coverslips, and for
electrophysiological experiments, on 35-mm culture dishes (Corning,
Cambridge, MA). Assays were performed 4872 h after transfection.
Measurements of Calcium Ion Concentration
For single-cell [Ca2+]i measurements,
cells were incubated at 37 C for 60 min with 2 µM fura-2
AM in phenol red- and ATP-free medium 199 with HBSS. The cells were
subsequently washed with Krebs-Ringer solution, and kept for at least
0.5 h in this medium before measurements. All experiments were
performed in the same medium. Coverslips with cells were mounted on the
stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany)
attached to the Attofluor Digital Fluorescence Microscopy System (Atto
Instruments, Rockville, MD). Cells were examined under a 40x oil
immersion objective during exposure to alternating 340- and 380-nm
light beams, and the intensity of light emission at 520 nm was
measured. The ratio of light intensities, F(340)/F(380), which reflects
changes in Ca2+ concentration, was simultaneously followed
in several single cells. Unless otherwise stated, all drugs were added
in 1-ml aliquots to reach the final working concentration.
Electophysiological Recordings
Electrophysiological experiments were performed on HEK293
cells at room temperature using whole-cell, gigaohm-seal recording
techniques (28). Voltage-clamp recordings were carried out using a
Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA)
and were filtered at 2 kHz using a lowpass Bessel filter. Patch
electrodes, fabricated from borosilicate glass (type 7740; World
Precision Instruments, Sarasota, FL) using a Flaming Brown horizontal
puller (P-87: Sutter Instruments, Novato, CA), were heat polished to a
final tip resistance of 4 to 5 megohms (M
). Before seal formation,
liquid junction potentials were canceled. After gigaohm seal formation
(>5 G
), pipette capacitance was neutralized, and the patch membrane
was ruptured using gentle suction (access resistance < 15 M
).
All current records were captured using the software package AxoScope
1.0 in conjunction with a Digidata 1200 A/D converter (Axon
Instruments). To isolate ICa, patch electrodes were filled
with a solution containing (in millimolar concentration): 120 NaCl, 20
tetraethylammonium-Cl, 10 HEPES, 10 EGTA (pH was adjusted to 7.2 with
NaOH), and the bath solution contained (in millimolar concentration):
120 NaCl, 2.6 CaCl2, 4.7 KCl, 0.7 MgSO4, 10
glucose, 10 HEPES (pH adjusted to 7.2 with NaOH). A 3 M KCl
agar bridge was placed between the bathing solution and the reference
electrode. All drugs were added using a rapid perifusion system.
Calculations
Where appropriate, the results were expressed as means ±
SEM. Concentration-response relationships were fitted to a
four-parameter logistic equation using a nonlinear curve-fitting
program, which derives the EC50 and Hills values
(Kaleidagraph, Synergy Software, Reading, PA). The statistical
significance of mono- and multiexponential fits was assessed according
to the "extra sum of squares" principle; P < 0.01
was considered significant (GraphPad Prism, GraphPad Software, San
Diego, CA).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Stanko Stojilkovic, National Institute of Child Health and Human Development/Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510. E-mail: stankos{at}helix.nih.gov
1 Supported by Japanese Society for the Promotion of Science in
Biomedical and Behavioral Research at NIH. 
Received for publication September 9, 1997.
Revision received February 11, 1998.
Accepted for publication March 12, 1998.
 |
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