Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

Extracellular nucleotides (ADP, ATP, UDP, and UTP) function as signaling molecules that mediate a variety of biological effects through a family of cell surface receptors known as P2 receptors. This family is divided into two groups: the ionotropic P2X receptors and the metabotropic P2Y receptors (Dubyak and el-Moatassim, 1993; Boarder et al., 1995; Boarder and Hourani, 1998; Ralevic and Burnstock, 1998). Currently, seven mammalian P2X receptors (P2X_1-7) and eight mammalian P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) have been cloned and functionally characterized (Ralevic and Burnstock, 1998; von Kugelgen and Wetter, 2000; Communi et al., 2001; Nicholas, 2001; Sak and Webb, 2002). P2X receptors function as nonselective cation channels with inwardly rectifying current-voltage relationships (Dubyak and el-Moatassim, 1993; Ralevic and Burnstock, 1998). Activation of these receptors with ATP or 2-methylthio-ATP (2MeS-ATP) produces membrane depolarization. P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptors couple to G_q/11 and activate phospholipase C, resulting in increased inositol phosphate-3 formation and mobilization of intracellular Ca²⁺ (Parr et al., 1994; Communi et al., 1995; Schachter et al., 1996; Lazarowski et al., 2001). The P2Y₁₁ receptor activates both phospholipase C and adenylyl cyclase (Communi et al., 1997), whereas P2Y₁₂ and P2Y₁₃ receptors are coupled solely to G_i and inhibition of adenylyl cyclase (Daniel et al., 1998; Hollopeter et al., 2001). P2Y₁₄ receptors are orphan G protein-coupled receptors that are activated by UDP-glucose and couple to the G_i/o class of G proteins (Chambers et al., 2000). The skate (s) P2Y receptor has 61 to 64% sequence similarity to the human (h) P2Y₁ receptor, is coupled to G_q/phospholipase C, and has a rank order of potency similar to the P2Y₁ receptor (Dranoff et al., 2000).

Previously, we showed that agonist activation of the P2Y₁ receptor expressed in Xenopus laevis oocytes stimulated a slowly activating inward current that inactivated within seconds after stimulation (O'Grady et al., 1996). The channel exhibits steady-state inactivation at strong hyperpolarizing potentials. This inward current was identified previously as the transient inward (T_in) current and was first observed after injection of mRNA from rat brain (Parker et al., 1985) and subsequently observed when cloned 5-hydroxytryptamine-1a and 5-hydroxytryptamine-2c receptors were expressed in oocytes (Ni et al., 1997). The channel is expressed in stage V and VI oocytes but seems to be absent in earlier stages of oocyte maturation. It is reversibly blocked by polyvalent cations including Ba²⁺, Mn²⁺, and La³⁺. T_in current activation requires membrane hyperpolarization and an increase in intracellular Ca²⁺ (Parker et al., 1985; Ni et al., 1997). Expression of G_q in mature oocytes was found to be sufficient for activation of the T_in current (Guttridge et al., 1995). The channel responsible for the T_in current has not been cloned and seems to represent a new family of ion channels that has not been previously characterized.

Interactions between expressed membrane proteins and endogenous ion channels that produce altered properties of these channels have been documented previously. For example, previous studies of Ca²⁺-activated Cl channels (CaCC) in bovine artery endothelial cells showed that biophysical properties of the channel could be modulated by expression of cystic fibrosis transmembrane conductance regulator (CFTR) (Wei et al., 2001). Stimulation of the cells with forskolin and 3-isobutyl-1-methylxanthine produced activation of CFTR and simultaneously inhibited ATP-dependent activation of endogenous CaCC activity. This effect of CFTR on the regulation of CaCC function was independent of the PDZ domain located at the C terminus of CFTR, but was shown to involve sequences within the R domain. CFTR has also been shown to modulate the function of amiloride-sensitive Na⁺ channels expressed in X. laevis oocytes (Boucherot et al., 2001). In this study, the first functional nucleotide-binding domain (NBF1) was proposed to be an interaction site between the two channels because mutations in the NBF1 region of CFTR resulted in a decrease in its ability to inhibit amiloride-sensitive Na⁺ channel activity.

In this study, we examined the effects of native P2Y receptor subtypes on channel function and observed that several members of the P2Y receptor family modified the functional properties of the channel. To address the hypothesis that these receptors modulate the gating and voltage dependence of the T_in channel through membrane-delimited interactions involving specific structural domains of the receptor, we constructed truncation mutants and chimeric receptors involving the C-terminal regions of hP2Y₁ and hP2Y₂ receptors and determined the effects of these mutations on the biophysical properties of the T_in channel. Our results indicate that the C-terminal domains of these P2Y receptors are involved in regulating voltage dependence or inactivation gating of the T_in channel. An analysis of C-terminal sequences of P2Y receptors suggests that there are protein-protein interaction domains, distinct from their PDZ-binding motifs, which are involved in the coupling and modulation of channel function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Materials. X. laevis frogs were purchased from Xenopus I (Ann Arbor, MI) and maintained in aquaria as suggested by the supplier. Collagenase and gentamicin were obtained from Invitrogen (Carlsbad, CA). 2MeS-ADP and 2MeS-ATP were obtained from Sigma/RBI (Natick, MA). UDP, UTP, carbachol, bradykinin, and isoproterenol were obtained from Sigma (St. Louis, MO). [D-Pen²,D-Pen⁵]-enkephalin was obtained from Bachem Biosciences (King of Prussia, PA).

Construction of Truncated and Chimeric Receptors. Truncated P2Y receptors were constructed by polymerase chain reaction using a 3' primer with a stop codon at the desired location and an XhoI restriction site to aid in subcloning. Chimeric receptors were generated by overlap-extension polymerase chain reaction (Ho et al., 1989). All receptor constructs were verified by sequencing and were subcloned into pcDNA3.

Preparation of RNA for Injection. cRNA was synthesized from linear cDNA encoding either wild-type P2Y receptor or mutants using Megascript (Ambion, Austin, TX).

Oocyte Isolation and Injection. Ovarian lobes from adult X. laevis frogs were removed from anesthetized animals under sterile condition. The tissue mass was dissociated with collagenase solution (90 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 10 mM HEPES, pH 7.4, and 250 units/ml collagenase). Stage V and VI oocytes were sorted, defolliculated, and maintained in modified Barth's saline solution (MBS solution: 90 mM NaCl, 2 mM KCl, 0.82 mM MgSO₄, 0.74 mM CaCl₂, and 10 mM HEPES, pH 7.4, supplemented with 0.05 µg/µl gentamicin) at 19 to 20°C. Oocytes were injected with cRNA transcripts (46 ng/oocyte) using a Nanoject oocyte injection system (Drummond Scientific (Broomall, PA). Control oocytes were injected with 46 nl of sterile water. Oocytes were stored for 2 to 7 days in MBS solution before analysis.

Peptide Synthesis and Purification. Peptides of 20 amino acids corresponding to the C-terminal sequence of the hP2Y₁ receptor (RKASRRSEANLQSKSEDMTL) or the hP2Y₂ receptor (RRSDRTDMQRIGDVLGSSED) were synthesized by the MicroChemical Facility at the University of Minnesota (St. Paul, MN). Peptides were purified by high-pressure liquid chromatography before injection, and the appropriate amino acid composition was confirmed by amino acid analysis.

Electrophysiological Measurements. Electrophysiological measurements were made using the two-electrode voltage-clamp technique at 20°C. Recordings were conducted in Cl-free MBS solution (90 mM NaMeSO₄, 2 mM KMeSO₄, 0.82 mM MgSO₄, 0.74 mM calcium gluconate, and 10 mM HEPES, pH 7.4). Electrodes were placed in a separate Cl-containing MBS solution and connected to the oocyte bathing solution with an agar bridge. Current- and voltage-measuring electrodes were pulled from borosilicate filament glass to resistances between 2 and 5 M when filled with 0.5 M KCl. Data acquisition and analysis was performed with a Pentium PC using pCLAMP 8 software (Axon Instruments, Inc., Union City, CA).

Analysis and Statistics. Statistical significance was determined using Student's t test. Statistical significance was accepted at p < 0.05. Conductance-voltage relationships were analyzed using a Boltzmann function (Y = 1/1 + exp(V₅₀  X/slope factor), where V₅₀ represents the voltage at which the conductance is half-maximal, slope factor represents the relative degree of voltage dependence (steepness of the curve), Y represents the normalized conductance, G/G_{140 mV}, and X represents a specific voltage.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

We previously reported that 2MeS-ADP stimulation of X. laevis oocytes expressing the hP2Y₁ receptor activated a voltage-dependent current with gating characteristics that were identical with the endogenous T_in channel in oocytes (O'Grady et al., 1996). The ability to measure these currents was dependent on receptor expression and on the presence of agonist. We followed up on these initial studies and report here that although all G_q-coupled P2Y receptors are capable of activating T_in channel currents in the presence of their cognate agonists, the electrophysiological properties of the channel vary markedly depending on the subtype of the receptor.

Bradykinin Activation of the hB₁-Bradykinin Receptor Elicits T_in Currents in X. laevis Oocytes. Figure 1A shows representative T_in current traces recorded from oocytes expressing the G_q-coupled hB₁-bradykinin receptor. Oocytes were held at 0 mV and then stepped to 140 mV and +80 mV in the presence of a 2 µM bradykinin. In the absence of either receptor mRNA or bradykinin, no time-dependent currents were observed upon hyperpolarization. In contrast, bradykinin elicited a characteristic T_in channel current in oocytes injected with hB₁-bradykinin receptor mRNA but not in noninjected oocytes (data not shown). Results for several G_q-coupled P2Y receptors were very similar, with the exception that a varying amount of T_in channel activation was observed under basal conditions in oocytes expressing P2Y receptors before agonist stimulation (O'Grady et al., 1996). This basal activation was probably caused by the accumulation in the bathing solution of nucleotides released from oocytes, a result similar to that observed in mammalian cells (Parr et al., 1994; Schachter et al., 1996).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Voltage dependence of the Tin current elicited by agonist-activated Gq-coupled receptors expressed in X. laevis oocytes. A, representative current traces recorded from oocytes expressing the hB1-bradykinin receptor. Oocytes were held at 0 mV and then stepped from 140 mV to +80 mV in 20-mV increments. The data represent peak inward current at each voltage step. The I-V relationship was fit with a Boltzmann function. The reversal potential of hB1-bradykinin receptor was determined from the fit and had a value of +13 mV. B, normalized conductance as a function of voltage for the Tin channel activated by hP2Y1 (n = 13), hP2Y4 (n = 9), rM1-muscarinic (n = 16), and hB1-bradykinin (n = 13) receptors. C, normalized conductance as a function of voltage for hB1-bradykinin (n = 13), hP2Y1 (n = 13), hP2Y11 (n = 15), and the sP2Y (n = 12) receptors. The V50 values and slope factors for each conductance are listed in Table 1. D, the effects of hP2Y1 receptor expression levels on the conductance-voltage relationship of the Tin channel; inset: conductance values obtained from oocytes injected with increasing amounts of hP2Y1 receptor mRNA (nanograms).

Voltage Dependence of the T_in Current Elicited by Agonist-Activated G_q-Coupled Receptors Expressed in X. laevis Oocytes. Figure 1B shows the normalized conductance-voltage relationships for G_q-coupled receptors expressed in X. laevis oocytes. Only G_q-coupled receptors elicited T_in channel currents, because agonist-activated ₂-adrenergic (50 µM isoproterenol; G_s-coupled), -opioid (5 µM [D-Pen²,D-Pen⁵]-enkephalin; G_i-coupled), M₂-muscarinic (10 µM carbachol; G_i-coupled), and P2Y₁₂ (20 µM 2MeS-ADP; G_i-coupled) receptors were unable to activate T_in currents after hyperpolarization (data not shown). The conductance-voltage curves were analyzed using a Boltzmann function, where V₅₀ represents the voltage at which the conductance was half-maximal (described under Materials and Methods). The V₅₀ value for the conductance activated by the hP2Y₁ receptor, but not the rat (r) M₁-muscarinic or hP2Y₄ receptor, was shifted markedly to a more negative voltage compared with the conductance activated by the hB₁-bradykinin receptor and was significantly different from the V₅₀ value elicited by the hB₁-bradykinin receptor (Table 1). Two other P2Y receptors, the hP2Y₁₁ and the sP2Y receptors, also activated currents with significantly more negative V₅₀ values than the currents activated by the hB₁-bradykinin or the rM₁-muscarinic receptor (Fig. 1C) (Table 1).

Role of the hP2Y₁ Receptor C Terminus in Modulating T_in Channel Voltage Sensitivity. The data presented above suggest that the hP2Y₁ and hP2Y₁₁ receptors not only activate T_in channels, but also modulate channel properties, possibly through direct protein-protein interactions. To test this hypothesis, we examined the conductance-voltage relationship of the T_in current elicited by a chimeric hB₁-bradykinin receptor in which the C-terminal domain was replaced by the C-terminal region from the hP2Y₁ receptor (hB₁/Y₁). Figure 2A shows that the conductance-voltage relationship of the T_in channel elicited by the hB₁/Y₁ chimeric receptor was essentially identical with that elicited by the activated hP2Y₁ receptor. These data demonstrate a "gain in function" of the hB₁-bradykinin receptor containing the hP2Y₁ receptor C-terminal domain and strongly suggest that the C-terminal region of the hP2Y₁ receptor is involved in regulating the properties of the T_in channel.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of C-terminal truncation on the conductance-voltage relationship. A, normalized conductance as a function of voltage for Tin currents elicited by activated hB1-bradykinin (n = 13), hP2Y1 (n = 13), and hB1/Y1 chimeric (n = 6) receptors. B, location of truncation mutations introduced into the hP2Y1 receptor. The last four amino acids, DTSL, represent a consensus class 1 PDZ-binding motif. C, normalized conductance as a function of voltage for Tin currents elicited by activated hB1-bradykinin (n = 13), hP2Y1 (n = 13), hP2Y1360tr (n = 17), and hP2Y1369tr (n = 10) receptors. D, normalized conductance as a function of voltage for Tin currents elicited by activated hP2Y1 (n = 13), hP2Y1334tr (n = 9), hP2Y1342tr (n = 18), and hP2Y1349tr (n = 9) receptors. The V50 values and slope factors for each conductance are listed in Table 1.

Role of the C-Terminal Domain of the hP2Y₂ and rP2Y₆ Receptors in T_in Channel Inactivation Gating. We also observed a marked difference in the inactivation gating of the T_in channel depending on which P2Y receptor was responsible for activating the current. Thus, whereas the current elicited by the hP2Y₁ receptor was almost completely inactivated within 3 s of the hyperpolarizing pulse, the current elicited by either the hP2Y₂ (stimulated with 40 µM UTP) or the rP2Y₆ receptor (stimulated with 40 µM UDP) showed significantly slower inactivation. (Fig. 3A). As observed with the voltage gating of the channel, the time course of inactivation was dependent on the identity of the C-terminal domain of the receptor. Thus, the inactivation time courses of the hP2Y₁ receptor containing the hP2Y₂ C-terminal domain (hY₁/Y₂ chimera) (Fig. 3A) or the hB₁-bradykinin receptor containing the hP2Y₂ C-terminal domain (hB₁/Y₂) (Fig. 3B) were identical with that of the wild-type hP2Y₂ receptor (Fig. 3A).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Inactivation gating of receptor-activated Tin currents. A, representative traces of the Tin conductance elicited by activated hP2Y1, rP2Y6, hP2Y2, and hY1/Y2 chimeric receptors. Tin channels were monitored for 5 s in the presence of the appropriate receptor agonist. UTP (40 µM) and UDP (40 µM) were used to stimulate the hP2Y2 and rP2Y6 receptors, respectively (Parr et al., 1994; Communi and Boeynaems, 1997). 2MeS-ADP (20 µM) was used to activate the hP2Y1 receptor and hY1/Y2 chimeric receptors. B, representative traces of currents elicited by agonist-activated hB1-bradykinin and hB1/Y1, and hB1/Y2 chimeric receptors after step hyperpolarization to 140 mV. C, Ifinal/Imax is the current amplitude at the end of the voltage pulse (as indicated by the open arrows in parts A and B) divided by the maximum inward current and represents the degree of Tin channel inactivation gating. This ratio was significantly increased in hP2Y2 (n = 10), rP2Y6 (n = 11), hY1/Y2 (n = 7), hY2/Y1 (n = 7), and hB1/Y2 (n = 7) chimeric receptors (p < 0.05).

C-Terminal Sequence Comparisons of P2Y Receptors. Figure 4 compares the C-terminal sequences of all five of the G_q-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁). Within the C-terminal domains of the hP2Y₁, hP2Y₁₁, and sP2Y receptor, all of which modulate the voltage activation of the T_in channel, a common sequence motif was observed (RRSEQXK/RSE) (bold letters identify conserved amino acids between P₂Y receptor subtypes). Importantly, this sequence motif falls within the narrow region of the hP2Y₁ receptor C-terminal domain shown to be involved in modulating voltage sensitivity (Fig. 2). Likewise, a different conserved sequence motif (QRXG/R) was observed in the C-terminal domains of the hP2Y₂ and rP2Y₆ receptor, both of which modulate T_in channel inactivation. This motif is not present in the other P2Y receptors.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   C-terminal sequence comparisons between P2Y receptors. The underlined and bolded amino acids represent potential sequences involved in protein-protein interactions with the Tin channel. hP2Y1, hP2Y11, and sP2Y receptors modulate voltage dependence, whereas hP2Y2 and rP2Y6 receptors modulate inactivation gating. A similar sequence motif affecting inactivation gating of the channel is present in the mouse (m) P2Y6 receptor. The boxed amino acids in the hP2Y1 and hP2Y2 receptor sequences indicate the peptide sequences used for experiments shown in Fig. 5.

Effects of Y1 and Y2 C-Terminal Peptides on the Conductance-Voltage Relationships and Inactivation Gating of the T_in Channel. To examine whether the unique sequence motif present in the C-terminal region of the hP2Y₁ receptor was able to modulate the conductance-voltage relationship of the T_in current, a synthetic peptide (Y1 peptide) (boxed region in Fig. 4) was injected into hP2Y₁342tr-expressing oocytes (final concentration  500 nM), and the conductance-voltage relationship of the T_in channel was determined (Fig. 5A). The V₅₀ value of the conductance elicited by the hP2Y₁342tr receptor with 500 nM Y1 peptide (72.3 mV) was significantly different from the value elicited by the hP2Y₁342tr receptor alone (V₅₀ = 49.0 mV) and similar to that elicited by the wild-type hP2Y₁ receptor (V₅₀ = 72.5 mV) (Table 1). The Y1 peptide produced a similar negative shift in the V₅₀ value (68.0 mV) in oocytes expressing the hB₁-bradykinin receptor compared with the hB₁-bradykinin receptor alone (43.0 mV) (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of Y1 and Y2 C-terminal peptides on the conductance-voltage relationship and inactivation gating of Tin currents. A, normalized conductance as a function of voltage of Tin currents elicited by the agonist-activated hP2Y1342tr receptor (Y1tr; n = 18), hP2Y1342tr receptor with 500 nM Y1 peptide (n = 8), and hP2Y1 receptor (n = 13). B, normalized conductance as a function of voltage of Tin currents elicited by the hB1-bradykinin receptor (n = 13), the hB1-bradykinin receptor with 500 nM Y1 peptide (n = 7), and the hP2Y1 receptor (n = 13). C, Ifinal/Imax ratios of the Tin current elicited by the indicated receptors were calculated as described in the legend to Fig. 3. The ratios were significantly increased for the hP2Y1342tr receptor with 500 nM Y2 peptide (n = 6), the hB1-bradykinin receptor with 500 nM Y2 peptide (n = 6), and the hP2Y2 receptor (n = 10) (p < 0.05).