Functional Role of Alternative Splicing in Pituitary P2X2 Receptor-Channel Activation and Desensitization

Taka-aki Koshimizu1, Melanija Tomic, 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo, 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.

 
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. 2Go). (The assigned accession numbers by GenBank for clones 3 to 6 are AF020756, AF020757, AF020758, and AF020759, respectively.)



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Figure 2.
 
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 1Go). 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. 3Go). 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. 3Go).


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Table 1. Deletion and Insertion Sequence Organization of P2X2R Splice Variants

 


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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.

 
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. 4AGo, 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. 4AGo, 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 (30–40%). 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. 2Go and 3Go). 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.

 
Subunit specific expression in GT1/P2X2R and GT1/P2X2-2R cells was confirmed by RT-PCR. Figure 1AGo 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. 1BGo). 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 4Go, 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. 4BGo). 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. 4CGo, left panel) and [Ca2+]i measurements (Fig. 4CGo, 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. 5Go). 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. 5BGo). Figure 5AGo 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.

 
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. 6AGo). 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 6BGo 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.



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Figure 6. Activation and Desensitization of P2X2R and P2X2-2R Expressed in GT1 Neurons

A, Activation of P2X2R and P2X2-2R expressed as the activation time (time needed to reach the maximum increase in [Ca2+]i subtracted for the delay in onset of the ATP response; n >= 5). The peak rates are half-maximal at {approx}51 µM and {approx}49 µM for P2X2R and P2X2-2R, respectively. B, Desensitization of wild-type and spliced channels. Experimental tracings from 10 representative cells are shown (dotted lines). In all cases, one exponential component is sufficient to describe the desensitization rate for both channels (solid line). The fitted function is extrapolated for clarity. The numbers above curves indicate the calculated rate constants of desensitization (k).

 
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. 7AGo). The calculated half-times for recovery of these two channels were comparable (Fig. 7BGo). 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 ({triangleup}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.

 
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. 8AGo, the first four fractions are enriched with gonadotrophs, fractions 5–9 with somatotrophs, and fractions 11–15 with lactotrophs. The transcripts for P2X2R were observed in somatotroph, but not in gonadotroph and lactotroph, fractions of anterior pituitary cells (Fig. 8BGo).



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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.

 
Somatotrophs exhibit spontaneous and extracellular calcium-dependent [Ca2+]i transients (Fig. 9Go), 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. 9AGo). Stimulatory effects of ATP on the frequency of [Ca2+]i transients were consistently observed in 5–100 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+.

 
Removal of extracellular Ca2+ abolished spontaneous [Ca2+]i transients. In such cells, ATP was unable to increase [Ca2+]i (Fig. 9BGo, 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. 9BGo, upper panel, +Ca2+). ATP, ATP{gamma}S, and 2 methylthio-ATP were found to be equipotent as agonists, whereas BzATP and ADP were less potent (Fig. 9BGo). Conversely, adenosine, AMP, {alpha},ß-methylene-ATP, ß,{gamma}-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. 6Go, 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. 10Go, 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.

 
Since the transcripts for P2X2R and P2X2-2R were equally abundant (Fig. 1Go) 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 10BGo 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 ({gamma}-aminobutyric acid and glycine channels). The second contains glutamate receptor channels and is composed of {alpha}-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 35–50% identity and 50–65% 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 {alpha},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 {alpha},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 3–6, 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 3–6 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Ham’s 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 Earle’s 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. 2Go. 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. 1Go, 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 48–72 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{Omega}). Before seal formation, liquid junction potentials were canceled. After gigaohm seal formation (>5 G{Omega}), pipette capacitance was neutralized, and the patch membrane was ruptured using gentle suction (access resistance < 15 M{Omega}). 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 Hill’s 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. Back

Received for publication September 9, 1997. Revision received February 11, 1998. Accepted for publication March 12, 1998.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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