Identification of Amino Acid Residues Contributing to Desensitization of the P2X2 Receptor Channel*

The P2X2 receptor (P2X2R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X2R transcript accounts for the existence of several transcript types, one of which (P2X2–2R) encodes a functional channel. P2X2–2R lacks a stretch of cytoplasmic C-terminal amino acids (Val370-Gln438) and exhibits rapid and complete desensitization, whereas P2X2R desensitizes slowly and incompletely. The role of the C terminus in P2X2R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1–7 neurons. Deletion studies indicated that the Arg371-Ile391 segment of the P2X2R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine. Only two of these replacement mutants, at Arg371-Thr372-Pro373and Lys374-His375-Pro376, had an enhanced rate of desensitization. Single amino acid deletions in the P2X2R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro373-Lys374-His375-Pro376, required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro373-Pro376 sequence of P2X2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.

P2X receptors (P2XR) 1 are ATP-gated cationic channels expressed in a variety of excitable cell types (1). Ligand binding to these channels activates an inward current, associated with an increase in the intracellular free Ca 2ϩ concentrations ([Ca 2ϩ ] i ) (2). The cDNA cloning of ionotropic ATP receptors identified seven subunits (P2X 1 R to P2X 7 R), each of which individually can form a cation-permeable pore in heterologous expression systems (3)(4)(5)(6)(7)(8)(9). These channels share a common hydrophobicity profile of two putative transmembrane domains (M1 and M2) connected with a large hydrophilic loop and intracellular N and C termini (10). There is 50 -65% amino acid sequence similarity overall between pairs of P2XR. In particular, the sequences from the N termini to the second transmembrane domains are relatively conserved, whereas the C-terminal tails display the least sequence similarity to each other (11). The physiological significance of the variations in the C-terminal structure has not been elucidated.
P2XR subtypes differ with respect to their ligand selectivity profiles, antagonist sensitivity, and desensitization rates (10,12). Based on differences in the desensitization kinetics, these channels can be divided into two groups: rapidly desensitizing channels (P2X 1 R and P2X 3 R) and slow desensitizing channels (P2X 2 R, P2X 6 R, and P2X 7 R) (11). P2X 4 R expressed in oocytes exhibits strong desensitization, but when expressed in HEK293 cells this channel desensitizes slowly (7,13,14). Two experimental approaches have been used to study the molecular mechanism underlying P2XR desensitization, the construction of chimeric channels between slowly and rapidly desensitizing subtypes and the co-expression of both types (5,15). Chimeric studies suggest that the responsible domains for desensitization are localized within the two transmembrane regions of P2X 1 R and P2X 3 R (15). Co-transfection of the expression plasmids for these two channel types was also found to yield a P2XR with altered desensitization and agonist selectivity properties, indicating that such channels are presumably heteropolymers (5). Recently, a new view about P2XR desensitization has emerged. The P2X 2 R splice variant, termed P2X 2-2 R or P2X 2b R (16 -18), lacks a stretch of the C-terminal amino acids of the P2X 2 R molecule (Val 370 -Gln 438 ) and encodes a functional channel that desensitizes faster than the wildtype. This observation suggests the importance of the spliced segment for prolonged Ca 2ϩ influx through wild-type channels. The P2X 2-2 R and several other splice variants were observed in neuronal and pituitary tissues (17,18). In pituitary somatotrophs, co-expression of spliced and wild-type P2X 2 R was shown to provide an effective mechanism for controlled Ca 2ϩ influx (18).
In this study, the structural elements in the P2X 2 R C terminus that are responsible for prolonged activation of wild-type channels were examined by generating diverse receptor mutants and monitoring [Ca 2ϩ ] i in transfected single cells. For this purpose, several experimental approaches were employed. Initially, a series of C-terminal truncated mutants were produced to narrow the region(s) needed for the slow desensitization pattern of P2X 2 R. Subsequently, triple alanine replacement and single amino acid deletion mutants were constructed to precisely identify the amino acid sequence that is critical for long lasting Ca 2ϩ signaling by wild-type channels. Finally, spliced amino acids from the C terminus of the wild-type channel were gradually added to the splice channel to regain the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: P2X 2 R, wild-type P2X 2 purinergic receptor channels; P2X 2-2 R, spliced form of P2X 2 R; GT1-7, immortalized gonadotropin-releasing hormone-secreting hypothalamic neurons; PCR, polymerase chain reaction. slow desensitizing pattern of Ca 2ϩ signaling in response to prolonged agonist stimulation. The results indicate that a polypeptide region containing Pro 373 , Lys 374 , His 375 , and Pro 376 residues is important for prolonged Ca 2ϩ influx in agonistoccupied wild-type channels.

MATERIALS AND METHODS
Construction of Mutant P2X 2 R-A 1.5-kilobase pair cDNA fragment of the P2X 2 R, obtained from rat pituitary by reverse transcription-PCR (18), was subcloned into pBluescript II vector (Stratagene, La Jolla, CA) in its XhoI/EcoRV site and used for PCR as a template to generate receptor mutants. For construction of C-terminal truncated receptors, in-frame premature stop codons were introduced in PCR primers at corresponding amino acid positions Ser 431 , Leu 414 , Pro 392 , and Arg 371 . For alanine-scanning mutagenesis, three contiguous amino acids were changed to alanine in the C-terminal portion starting from Arg 371 to Ile 391 . New restriction sites for PstI were engineered to identify alanine mutant clones. A single amino acid deletion from the wild-type sequence and gradual additions of amino acids, which were spliced out from the wild-type, to the variant C terminus, were also generated by PCR. All PCR fragments for construction of mutant receptors were sucloned into pBluescript II and sequenced by the dideoxy chain termination method using Sequenase version 2.0 (Amersham Pharmacia Biotech). After confirming whole sequences of the PCR products, these mutations were cut with ClaI and XbaI and transferred to the ClaI/ XbaI site of the P2X 2 R expression vector, pME/P2X 2 R (18), to substitute corresponding sequences.
Cell Cultures and Expression Studies-Cell culture and transfection of the GT1-7 neurons were performed as described previously (18). Briefly, GT1 neurons were cultured in Dulbecco's modified Eagle's Medium and Ham's F12 medium (1:1) supplemented with 10% fetal calf serum and 100 g/ml ampicillin. On the day of transfection, 3 g of the plasmid DNA was mixed with 7 l of LipofectAMINE TM in 3 ml of serum-free OPTI-MEM medium (Life Technologies, Inc.), incubated for 20 min at room temperature, and then applied to cells (10 6 cells/60-mm dish). After 6 h of incubation, the medium was replaced with fresh culture medium and cells were allowed to grow for 24 h. For single cell calcium recordings, transfected cells were plated on poly-L-lysine-treated coverslips. Assays were performed 48 h after transfection.
Measurements of Calcium Ion Concentration-For single cell [Ca 2ϩ ] i measurements, cells were incubated at 37°C for 60 min with 2 M fura-2/AM in phenol red-and ATP-free Dulbecco's modified Eagle's medium. The cells were subsequently washed with Krebs-Ringer solution containing 1.2 mM Ca 2ϩ and kept for at least ½ h in this medium prior to measurements. All experiments were performed in cells bathed in Krebs-Ringer solution containing 1.2 mM Ca 2ϩ and 100 nM nifedipine at room temperature. Nifedipine was added to exclude the participation of non-inactivating L-type Ca 2ϩ channels, which are expressed in GT1-7 cells (19). 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 40ϫ 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 Ca 2ϩ concentration, was simultaneously followed in several single cells.
Calculations-The statistical significance of mono-and multiexponential fits were assessed according to the "extra sum of squares" principle; p Ͻ 0.01 was considered significant (GraphPad Prism, Graph-Pad Software, San Diego, CA). The results for desensitization rate were expressed as means Ϯ S.E. Student's t-test was used for statistical comparison among means where applicable. Differences in a p value less than 0.01 were considered significant. In all figures showing [Ca 2ϩ ] i changes, the tracings are normalized with respect to the maximum in the amplitude (100%).

RESULTS AND DISCUSSION
The presence of multiple splice variants of P2X 2 R in various cell types has been reported by several groups (16 -18). One of these transcripts, P2X 2-2 R, forms a functional channel when expressed in Xenopus oocytes, GT1-7, and HEK293 cells. In our study, the immortalized GT1-7 neurons were used as an expression system because of the following three reasons. First, these cells express neither P2Y calcium-mobilizing receptors nor P2X receptor channels, as documented by the inability of 100 M ATP to rise [Ca 2ϩ ] i in cells bathed in Ca 2ϩ -deficient as well as Ca 2ϩ -containing medium. Second, the pattern of single cell [Ca 2ϩ ] i responses and the profiles of isolated Ca 2ϩ currents sufficiently demonstrated a marked difference in the desensitization kinetics between wild-type and splice variant subunits (18). Since the rates of desensitization for these channels were comparable in both Ca 2ϩ current and [Ca 2ϩ ] i measurements, here we employed only [Ca 2ϩ ] i measurements. Finally, our preliminary experiments for RNA and protein levels confirmed that further splicing of RNA transcripts for the wild-type channel molecule does not occur in these neuronal cells, allowing channel subunit-specific characterization.
The P2X 2-2 R lacks a stretch of C-terminal amino acids (Val 370 -Gln 438 ), indicated by dashed lines in Fig. 1A. Comparison between the wild-type and spliced channels suggested that they do not differ in terms of their activation properties, EC 50 values to ATP, maximum current/Ca 2ϩ responses to ATP, or The amino acids of P2X 2 R indicated by the arrows are changed to in-frame stop codons. The resulting P2X 2 R mutants were transfected in GT1-7 neurons, and Ca 2ϩ influx was measured using the Ca 2ϩ indicator, fura-2/AM. Residues within the gray area contain a critical region for sustained Ca 2ϩ influx. B, patterns of [Ca 2ϩ ] i responses evoked by 100 M ATP in GT1-7 neurons expressing the truncated mutant channels, as well as the wild-type and spliced channels. In these and the following experiments, at least three independent trials were performed, with at least 30 single cells analyzed in each trial. Representative tracing from those trials (dotted lines), with normalized amplitudes of [Ca 2ϩ ] i responses (Y axes) are shown. In all cases, one exponential component is sufficient to describe the desensitization rate (solid lines). The fitted function is extrapolated for clarity. The numbers above each curve indicate the calculated desensitization rate constants (k) expressed as means Ϯ S.E. the recovery times from desensitization (18). They did, however, exhibit different rates of desensitization (Fig. 1B). The spliced channels P2X 2-2 R desensitized relatively rapidly and completely (within 2-3 min), whereas the wild-type channels desensitized slowly and incompletely (16 -18). Thus, the presence of the Val 370 -Gln 438 sequence in wild-type channels is critical for their slow desensitization.
To elucidate the functional role of the C-terminal tail in control of the rate of desensitization, four truncated P2X 2 R were constructed and expressed in GT1-7 neurons (Fig. 1A). Removal of the putative cytoplasmic tail up to Pro 392 resulted in mutant receptors that lost the last 81 amino acids of P2X 2 R and did not alter the pattern of [Ca 2ϩ ] i responses to 100 M ATP (Fig. 1B, three central tracings). This argues against the potential importance of the proline-rich segment (20), observed only within the P2X 2 R subunits, in activation and desensitization of these channels. In contrast, removal of the polypeptide sequence to Arg 371 resulted in desensitization rates that were indistinguishable from those observed in P2X 2-2 R (Fig. 1, two  bottom tracings). The truncated channel also showed a significant reduction in the amplitude of Ca 2ϩ response to 100 M ATP ([Ca 2ϩ ] i (ratio): P2X 2 R ϭ 3.12 Ϯ 0.24 (n ϭ 47) versus P2X 2 R-Arg 371 ϭ 1.42 Ϯ 0.18 (n ϭ 18)), indicating that the activation properties of this truncated channel were also altered. Since the first 69 amino acids in this segment are missing in the spliced channels, these results also indicate that the C-terminal tail of the P2X 2-2 R can substitute for the Arg 371 -Ile 391 segment but only in a transient activation of these channels.
To localize the C-terminal region responsible for the desensitization properties of the wild-type channel, specific amino acid(s) in the Arg 371 -Ile 391 segment were mutated, whereas the residual part of the C-terminal was retained. For this purpose, we used the alanine-scanning mutagenesis method and constructed seven mutant channels, in which three contiguous amino acids were substituted for triple alanine residues: RTP 373 /AAA, KHP 376 /AAA, SSR 379 /AAA, WPV 382 /AAA, TLA 385 /AAA, LVL 388 /AAA, and GQI 391 /AAA ( Fig. 2A). All constructs were functional in terms of the portion of cells expressing channels and the amplitudes of [Ca 2ϩ ] i in response to 100 M ATP (not shown). The last five mutant channels (SSR 379 / AAA, WPV 382 /AAA, TLA 385 /AAA, LVL 388 /AAA, and GQI 391 / AAA) also showed no difference in the rate of desensitization when compared with wild-type channels. In contrast, RTP 373 / AAA and KHP 376 /AAA mutants had a significant (p Ͻ 0.01) increase in the rate of desensitization compared with wild-type channels (Fig. 2, B and C). Although the RTP 373 /AAA channel resulted in a faster decline in [Ca 2ϩ ] i than the KHP 376 /AAA channel, neither rate of desensitization for these mutant channels fully reached that of the spliced channels (Fig. 2, B and C). This result indicates that the critical amino acids for development of a slow desensitizing pattern of Ca 2ϩ signaling by P2X 2 R are located within the C-terminal segment Arg 371 -Pro 376 (Fig. 2A, gray area).
When the amino acid sequences for the seven subunits of the P2XR family were aligned according to their hydrophobicities, the putative cytoplasmic C termini show very little similarity in terms of amino acid moiety and length (11). However, the polypeptide sequence Arg 371 -Pro 376 of P2X 2 R contains three conserved residues, Arg 371 , Pro 373 , and Lys 374 , among four members of slow desensitizing receptors, P2X 2 R, P2X 5 R, P2X 6 R, and P2X 7 R (Fig. 3A). These residues are not present in P2X 4 R, which desensitizes slowly when expressed in HEK293 cells (21), but rapidly when expressed in Xenopus oocytes (13,14). Furthermore, threonine at position 372 in the P2X 2 R tail is a potential phosphorylation site that can be modified by protein kinase C, (R/K)XX(T/S)X(R/K), or type II calmodulin-dependent kinase, (R/K)XX(T/S) (X represents any amino acid). To address the role of the conserved residues, as well as the Thr 373 residue, in the desensitization pattern of the wild-type channel, mutant receptors with single amino acid deletions from the wild-type C-terminal were compared with the splicing variant P2X 2-2 R or wild-type receptor. Elimination of four individual residues in this segment indicated the importance of two amino acids, Pro 373 and Lys 374 , in the desensitization pattern of wild-type channels. As shown in Fig. 3, B and C, both mutants exhibited an enhanced rate of desensitization when compared with wildtype channels, but neither channel alone was able to mimic the pattern of desensitization of the spliced channel. On the other hand, deletions of Arg 371 or Thr 372 resulted in no detectable change in the desensitization rate.
The functional properties of the P2X 2 R C-terminal were further analyzed by mutagenesis studies, in which amino acid residues spliced out from the wild-type tail were gradually added back to the splice site at the P2X 2-2 R C terminus (Fig.  4A). The common 34 amino acid end for P2X 2 R and P2X 2-2 R was also kept in these mutant receptors. In all mutants, the amplitudes of [Ca 2ϩ ] i in response to 100 M ATP were comparable with that of the wild-type channel. Additions of three amino acids in P2X 2-2 ϩ V-T 372 to the splice site did not result in an apparent change in the desensitization rate. Additional insertions of four amino acids from the wild-type sequence resulted in a gradual decrease in the rate of desensitization when compared with that of the spliced channels (P2X 2-2 ϩ V-P 373 , P2X 2-2 ϩ V-K 374 , P2X 2 ϩ V-H 375 , and P2X 2 ϩ V-P 376 in Fig. 4, B and C). These data indicate that insertion of the Pro 373 -Lys 374 -His 375 -Pro 376 sequence is required for sustained Ca 2ϩ influx. Thus, it is likely that the conserved Pro 373 and Lys 374 residues comprise a part of the functional region that is critical for development of the slow desensitization pattern of P2X 2 R.
These results indicate that desensitization rates in [Ca 2ϩ ] i responses following activation of wild-type, spliced, and mutant P2X 2 R channels correlate with the C-terminal structures of expressed channels. Earlier studies have shown comparable rates of desensitization for wild-type and spliced channels in isolated Ca 2ϩ current and [Ca 2ϩ ] i measurements (18), as well as in total current measurements (16). This does not exclude the need for current measurements through these non-selective cation channels and their mutants in future experiments. However, control of Ca 2ϩ influx through P2X 2 R channels is physiologically important because this ion represents an intracellular messenger that regulates a number of cellular functions, including plasma membrane excitability, hormone secretion, de novo protein synthesis, and apoptosis (22).
In this respect, the wild-type channel induces a prolonged, high amplitude Ca 2ϩ signal, similar to that observed in apoptotic cells (23). Conversely, deletion of the Pro 373 -Lys 374 -His 375 -Pro 376 sequence as a consequence of alternative splicing generates a channel capable of limiting Ca 2ϩ influx into the cells during prolonged agonist activation. Furthermore, the co-expression of wild-type and spliced channels provides an effective mechanism to sustain Ca 2ϩ signaling but protects the cells from overloading with Ca 2ϩ (18). For example, pituitary somatotrophs express both spliced and wild-type channel subunits, resulting in rapid but incomplete desensitization. This leads to the biphasic pattern of Ca 2ϩ signaling, which is com- FIG. 3. Effects of single amino acid deletions in the C terminus of P2X 2 R on calcium influx. A, the amino acid sequence of the cytoplasmic C-terminal part of the P2X 2 R is aligned with other members of the P2XR subunits (11). Conserved positions among the four members of slowly desensitizing channels are indicated by shading. P2X 4 R is also a slow desensitizing channel when expressed in HEK 293 cells but does not contain these residues. B, ATP (100 M) induced Ca 2ϩ responses of wild-type channels, spliced P2X 2-2 R, and four single amino acid deletion mutants. C, mean Ϯ S.E. values of the desensitization rates for these channels.  Fig. 1A), are identical with that of P2X 2 R and are followed by a common 34-amino acid C-terminal end. M2 refers to the putative second transmembrane domain. B, the patterns of ATP (100 M)-induced Ca 2ϩ influx through wild-type, spliced variant, and seven mutant channels. C, mean Ϯ S.E. values of the desensitization rates for these channels. posed of an early spike phase and sustained plateau phase (18). The probable heteromeric assembly of wild-type P2X 2 R with rapidly desensitizing P2X 3 R may also compose a channel with controlled cationic influx (24), but this mechanism is potentially available only for cells that express both subunits.
In conclusion, the presence of specific residues in the C terminus of P2X 2 R, with two conserved amino acids contributing significantly to the development of a sustained Ca 2ϩ influx through these channels, may indicate a common mechanism of desensitization for the P2XR family. The Lys 375 residue is common to four slow desensitizing channels, P2X 2 R, P2X 5 R, P2X 6 R, and P2X 7 R, whereas Pro 374 is only present in three of them, P2X 2 R, P2X 6 R, and P2X 7 R. On the other hand, the rapidly desensitizing P2X 1 R and P2X 3 R do not contain these two amino acids (11). These residues are also not present in P2X 4 R, a channel that desensitizes rapidly when expressed in oocytes (13,14). The removal of the Pro 373 -Pro 376 segment by splicing resulted in the rapidly desensitizing P2X 2-2 R (16 -18). Certainly, future experiments with insertion of this segment into rapidly desensitizing channels and its deletion from slow desensitizing P2XR will provide important insight into the functional regulation of P2XR by their C termini.