Introduction

Nucleotides, including ATP and UTP, are released from most cells and function as extracellular signaling molecules in a variety of cell types. Their biological effects are mediated through P2 receptors, which are divided into two classes: Ionotropic P2X receptors and metabotropic P2Y receptors [1–4]. Eight mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized functionally [4–9]. P2Y1, P2Y2, P2Y4, and P2Y6 receptors are known to couple solely to activation of phospholipase C through Gq/11 (and possibly Gi) resulting in production of IP3 and mobilization of intracellular Ca2+ [10–13]. The sP2Y receptor, which has 61%–64% sequence similarity to the hP2Y1 receptor, activates Gq/phospholipase C with similar rank nucleotide selectivity as that of the hP2Y1 receptor [14]. The hP2Y11 receptor activates both phospholipase C and adenylyl cyclase [15], whereas P2Y12, P2Y13, and P2Y14 receptors are coupled exclusively to Gi and inhibition of adenylyl cyclase [5, 7, 16].

Agonist activation of the hP2Y1 receptor expressed in Xenopus oocytes stimulated a previously characterized transient inward (Tin) current [17, 18]. The channel responsible for the Tin current has not been cloned, but appears to represent a new family of ion channels that has not been previously characterized at the molecular level. The Tin current was initially identified following injection of mRNA from rat brain [19] and subsequently observed when cloned 5-HT1a and 5-HT2c receptors were expressed in oocytes [20]. Tin activation required both membrane hyperpolarization and an increase in intracellular calcium in addition to agonist activation of these receptors [19, 20]. The Tin channel can be distinguished from endogenous Ca2+ and hyperpolarization-activated Cl− channels in Xenopus oocytes by its inactivation gating which occurs within 4 s following hyperpolarization [21]. Moreover, expression of Gαq in stage V and VI oocytes was found to be sufficient for activation of the Tin current, thus demonstrating a role for Gαq in regulation of this channel [22].

We showed recently that P2Y receptors are capable of activating and modulating the voltage dependence and inactivation of the Tin channel through interactions involving the C-terminal domains of these receptors that was independent of PDZ-binding motifs [18, 23]. In the present study, we investigated the role of TM3 of the hP2Y1 receptor in modulation of the Tin channel. Our results indicated that activation and Zn2+ sensitivity were dependent on the subtype of P2Y receptor expressed and that mutations within TM3 of the hP2Y1 receptor significantly altered these properties of the channel.

Materials and methods

Materials

Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, Michigan, USA) and maintained in aquaria as suggested by the supplier. Collagenase and gentamicin were obtained from Invitrogen (Carlsbad, California, USA). 2Methylthio-ADP (2MeS-ADP) and 2Methylthio-ATP (2MeS-ATP) were obtained from Research Biochemicals (Natick, Massachusetts, USA). Carbachol and bradykinin were obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

Site-directed mutagenesis

Site-directed mutagenesis was done with the Altered Sites in vitro mutagenesis system kit from Promega (Madison, Wisconsin, USA) following the manufacturer’s instructions.

Oocyte isolation and cRNA injections

Ovarian lobes from adult X. laevis frogs were removed from anesthetized animals under sterile conditions and the tissue mass was dissociated with collagenase solution (in mM: 90 NaCl, 1 KCl, 0.82 MgSO4, 10 HEPES (pH 7.4), 250 U/ml collagenase). Stage V and VI oocytes were sorted, defolliculated and maintained in modified Barth’s saline solution (MBS solution; in mM: 90 NaCl, 2 KCl, 0.82 MgSO4, 0.74 CaCl2, 10 HEPES, pH 7.4, supplemented with 0.05 µg/µl gentamicin) at 19–20 °C. cRNA was synthesized using the Ambion Megascript Kit (Austin, Texas, USA) from linear cDNA encoding the hB1-bradykinin receptor, wild-type P2Y receptors or mutants of the hP2Y1 receptor. Oocytes were injected with cRNA transcripts (46 ng/oocyte) using a Drummond Nanoject oocyte injection system. Control oocytes were injected with 46 nl of sterile water. Oocytes were stored for 2–7 days in MBS solution.

Electrophysiological measurements

Electrophysiological measurements were made using the two-electrode voltage clamp technique at 20 °C. Recordings were performed in Cl−-free MBS solution (in mM): 90 NaMeSO4, 2 KMeSO4, 0.82 MgSO4, 0.74 CaGluconate, 10 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 MΩ when filled with 0.5 M KCl. Data acquisition and analysis was performed using pCLAMP 8 software (Axon Instruments, Union City, California, USA).

Agonist stimulation of expressed receptors

Agonist concentrations used for these experiments were selected to produce maximum receptor activation, based on previous concentration-response studies on P2Y receptors expressed in Xenopus oocytes [17]. Agonists were first added to the bathing solution to activate the receptors and then the membrane potential was stepped from 0 to −140 mV to activate the Tin channel. Maximum current responses to the voltage step protocol were achieved within 5 min after agonist addition to the bathing solution. 2MeS-ADP (20 µM) was used to stimulate hP2Y1, mutants of hP2Y1, and sP2Y receptors. 2MeS-ATP (40 µM) was used to stimulate hP2Y11 receptors. Bradykinin (2 µM) and carbachol (10 µM) were used to stimulate hB1-bradykinin and rM1-muscarinic receptors, respectively.

Analysis and statistics

Statistical significance was determined using Student’s t-test. Statistical significance was accepted at P-values <0.05. Conductance-voltage relationships were analyzed using a Boltzmann function Y = 1/1 + exp(V 50 − X/slope factor), where V 50 represents the voltage at which the conductance is half-maximal, slope factor represents the degree of voltage dependence (steepness of the curve), Y represents the normalized conductance, G/G −140 mV, and X represents a specific voltage). IC50 values were calculated using four-parameter logistic function (Y = 100/(1 + exp (log IC50 − X) × slope)), where X is the logarithm of concentration and Y is the response.

Results and discussion

Tin channel activation following stimulation of Gq coupled receptors expressed in Xenopus oocytes

Time-to-peak measurements were used as an indicator of the activation time course and were defined as the time between the capacitance current and the peak inward current (Figure 1A). It is important to note that the time-to-peak measurement does not reflect the time course of agonist binding or receptor activation, but instead is a measure of the time course of channel opening following the voltage step. When the hP2Y1 receptor and sP2Y receptor were expressed in Xenopus oocytes, the time-to-peak activation of the Tin channel at all voltages tested was significantly reduced compared to oocytes expressing the hB1-bradykinin receptor or Gqα subunit (Figure 1B). In contrast, when either rM1-muscarinic, hP2Y4, or hP2Y11 receptors were expressed in oocytes, time-to-peak values of the Tin channel were similar to those of hB1-bradykinin receptor or Gqα subunit but significantly different from hP2Y1 or sP2Y receptors (Figure 1C). These results indicated that hP2Y1 and sP2Y receptors were capable of decreasing the time interval between the initiation of membrane hyperpolarization and the beginning of Tin channel opening compared to other Gq coupled receptors, including other subtypes of P2Y receptors and the Gqα subunit. Expression of hP2Y1 or sP2Y receptors did not affect the rate of channel opening, as reflected by the slope of the current trace between the point of initiation and the peak inward current, compared to the hB1-bradykinin receptor (data not shown). These findings suggest that, unlike hB1-bradykinin or rM1-muscarinic receptors, an interaction occurs between the hP2Y1 (or sP2Y) receptor and the Tin channel that influences the ability of the channel to respond to membrane hyperpolarization.

Figure 1
figure 1

Effects of expressed Gq coupled receptor stimulation on hyperpolarization-induced activation of the endogenous Tin channel in Xenopus oocytes. A) Representative trace of the Tin channel current at −140 mV following stimulation of the expressed hP2Y1 receptor. Time-to-peak current measurements were used to assess hyperpolarization-induced activation of the channel and are defined as the elapsed time following the capacitance current to the peak inward current. B) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), Gq alpha subunit (n = 13), hP2Y1 receptor (n = 14), and sP2Y (n = 13). C) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), rM1-muscarinic (n = 12), hP2Y4 (n = 12), and hP2Y11 (n = 13) receptors.

Full size image

Effect of C-terminal domain truncation

We showed previously that the C-terminal domain of the P2Y1 receptor was involved in modulating the voltage dependence of activation of the Tin channel in Xenopus oocytes [18]. To determine whether the C-terminal domain of the hP2Y1 receptor was also involved in modulating activation, we compared the time-to-peak currents of the Tin channel in oocytes expressing either the wild-type hP2Y1 receptor or one of several C-terminal truncation mutants of the hP2Y1 receptor (Figure 2A). As shown in Figure 2B, the time-to-peak values of the Tin channel in oocytes expressing any of the truncation mutants were not significantly different from the values derived from oocytes expressing the wild-type hP2Y1 receptor, suggesting that the C-terminal domain was not involved in modulating activation of the channel.

Figure 2
figure 2

Effect of C-terminal domain truncation on channel activation. A) The relative locations of TM3 mutations (hP2Y1-H132A, hP2Y1-H132D, and hP2Y1-H132F) and C-terminal truncation sites within the hP2Y1 receptor are indicated. B) Comparison of time-to-peak measurements for hB1-bradykinin (n = 13), hP2Y1 (n = 14), hP2Y1334tr (n = 9), hP2Y1342tr (n = 15), hP2Y1349tr (n = 11), hP2Y1360tr (n = 15), and hP2Y1369tr (n = 13) receptors as the function of voltage.

Full size image

Effect of the TM3 mutations on activation and conductance-voltage relationships

The P2Y1 receptor contains a unique histidine residue at position 132 that is not present in other P2Y receptor subtypes. To determine if TM3 of the hP2Y1 receptor is important in modulating activation, we mutated His-132 to alanine (hP2Y1-H132A), aspartic acid (hP2Y1-H132D), and phenylalanine (hP2Y1-H132F) (Figure 2A), expressed the mutants in Xenopus oocytes, and determined the time-to-peak value of the Tin current following receptor activation. Time-to-peak current measurements of all TM3 point mutations were significantly increased compared to the wild-type hP2Y1 receptor (Figure 3A). Mutation of His-132 to alanine, aspartic acid, or phenylalanine increased time-to-peak current values compared to the wild-type hP2Y1 receptor, suggesting that the primary structure of TM3 plays a role in modulating channel activation. It is worth noting that TM3 of the hP2Y1 receptor is known to be important in agonist recognition [24–26]. The H132A mutation was previously shown to produce a shift in the 2MeS-ADP EC50 for phospholipase C activation from 1.94 ± 0.8 to 17.7 ± 4.7 nM when expressed in COS-7 cells [26]. To ensure full activation of the H132 mutant receptors, the 2MeS-ADP concentration used in these experiments was set at 20 µM.

Figure 3
figure 3

Effect of third transmembrane domain mutations on activation and conductance voltage relationships. A) Time-to-peak measurements for hB1-bradykinin (n = 13), wild-type hP2Y1 (n = 14), hP2Y1-H132A (n = 5), hP2Y1-H132D (n = 7), hP2Y1-H132F (n = 10), and hP2Y1-Q307K (n = 7) receptors as a function of voltage. B) Normalized conductance-voltage relationships for hB1-bradykinin (n = 13), wild-type hP2Y1 (n = 13), hP2Y1-H132A (n = 15), hP2Y1-H132D (n = 6), and hP2Y1-H132F (n = 6). The V 50 values and slope factors for each conductance are listed in Table 1.

Full size image

We also analyzed the conductance-voltage properties of the Tin channel currents activated by the mutant receptors. As shown in Figure 3B, the conductance-voltage relationships for these mutants were not significantly different from the wild-type hP2Y1 receptor. The V 50 values, which are defined as the voltages at which the current is one-half of that at −140 mV (see Materials and methods for more details), for each of the hP2Y1 receptor mutants were similar to the V 50 value of wild-type hP2Y1 receptor, but were significantly more negative than the V 50 value measured for the hB1-bradykinin receptor (Figure 3B, Table 1). These data demonstrated that the point mutations in TM3 of the hP2Y1 receptor did not modulate the voltage sensitivity of the Tin channel, as was previously shown for the C-terminal domain of the receptor [18, 23].

Table 1 V50 and slope factors of Tin currents elicited by agonist-activated receptors.
Full size table

Effects of Zn2+ on the inward current elicited by step hyperpolarization to −140 mV

Divalent cations have been shown previously to block Tin currents, with Zn2+ being the most potent of these blockers [17, 19]. Therefore, ZnCl2 was used to compare divalent ion sensitivity of the Tin current activated by hB1-bradykinin and P2Y receptors. Figure 4A shows the current response at −140 mV of the Tin channel elicited by agonist-activated hP2Y1 receptors in the presence of increasing concentrations of ZnCl2. The amplitudes of the Tin currents were reduced in a concentration-dependent manner in the range of 1 to 500 µM ZnCl2. Curves were analyzed by using a four-parameter logistic function as described in Methods. The IC50 value of ZnCl2 at the Tin channel activated by the hB1-bradykinin receptor was 65 µM, which was not significantly different from IC50 values obtained following activation by the Gqα subunit (98 µM; Figure 4B, Table 2) or hP2Y11 receptor (83 µM; Figure 4C, Table 2). It is worth noting that the inhibition of Tin channel current by ZnCl2 in oocytes expressing the Gqα subunit indicated that the effect of Zn2+ was not on receptor-mediated regulation of the Tin channel. In contrast, the IC50 of ZnCl2 for inhibition of Tin currents activated by the hP2Y1 receptor was significantly higher (Figure 4C, Table 2). The Zn2+ IC50 for inhibition of the currents elicited by the sP2Y receptor, which also has a His residue at position 132, was essentially identical to that of hP2Y1 receptor.

Figure 4
figure 4

Effects of Zn2+ on the inward currents elicited by step hyperpolarization to −140 mV. A) Representative current traces showing Tin currents activated by hP2Y1 receptor in the presence of 20 µM 2MeS-ADP. Agonist-stimulated inward current was inhibited by ZnCl2 in a concentration-dependent manner. B) Concentration-response relationship for Gq (n = 7) and hB1-bradykinin (n = 5). C) Inhibition of hP2Y1 (n = 6), sP2Y (n = 5), and hP2Y11 (n = 5) receptor-elicited Tin currents by [Zn2+]. The IC50 values are listed in Table 2.

Full size image
Table 2 IC50 values from ZN2+ concentration-response relationship in Figure 4.
Full size table

To investigate whether His-132 is important in modulating the Zn2+ sensitivity of the Tin current, we examined the effect of hP2Y1-H132A and hP2Y1-H132F mutations in the hP2Y1 receptor on Zn2+ sensitivity of the channel (Figure 5A). The Zn2+ IC50 values for currents elicited by either the hP2Y1-H132A or the hB1-bradykinin receptor were similar, whereas the IC50 value of Zn2+ for currents elicited by wild-type hP2Y1 receptor was significantly higher. In contrast, the hP2Y1-H132F receptor-activated current was considerably more sensitive to Zn2+ compared to wild-type hP2Y1 and the hP2Y1-H132A receptors (Figure 5A, Table 2), suggesting that mutations in the TM3 alter both Zn2+ sensitivity of the Tin channel and activation. A significant change in the slope of the concentration-response relationship was also observed following stimulation of the hP2Y1-H132F receptor. This change in slope reflected an increase in Zn2+ sensitivity of the channel and a dramatic shift in the threshold for Zn2+ inhibition of the channel. In contrast, C-terminal truncation nor the hP2Y1-Q307K mutation (an amino acid substitution in TM7) had no effect on Zn2+ sensitivity of the Tin channel (Figure 5B).

Figure 5
figure 5

Effects of Zn2+ on the inward currents elicited after stimulation of mutant P2Y1 receptors. A) Inhibition of hP2Y1 (n = 6), hP2Y1-H132A (n = 3) and hP2Y1-H132F (n = 5) receptor-elicited Tin currents by [Zn2+]. B) Inhibition of hP2Y1 (n = 6), hP2Y1342tr (n = 9), and hP2Y1-Q307K (n = 5) receptor-elicited Tin currents by [Zn2+]. The IC50 values are listed in Table 2.

Full size image

Taken together, the results of this study and our previous work [18] suggest the hypothesis that hP2Y1 and sP2Y receptors interact with the Tin channel at multiple sites, including their C-terminal tails and TM3 regions. Whereas the C-terminal tails appeared to be involved in voltage dependence and inactivation kinetics of the Tin channel, the TM3 region appeared to affect the response of the channel to membrane hyperpolarization and ZnCl2 sensitivity. The point mutations in TM3 might alter a direct interaction of the receptor with the channel or indirectly affect coupling between the receptor and the channel at a site other than the TM3 domain through changes in receptor tertiary structure. It is clear from these studies, however, that the C-terminal domain of the receptor does not influence hyperpolarization-induced activation or Zn2+ sensitivity. Moreover, activation and Zn2+ sensitivity were not affected by expression of the hP2Y11 receptor, even though this receptor subtype was shown previously to produce a shift in the voltage dependence of the channel similar to the hP2Y1 and sP2Y receptors [18]. This observation is consistent with the idea that the C-terminal region and TM3 domains of the receptor independently affect distinct functional properties of the Tin channel.

Analysis of the hP2Y11 receptor sequence showed that the corresponding residue to His-132 in TM3 is Thr-107. It is interesting to note that the Zn2+ IC50 values for the Tin currents elicited by hP2Y11, hP2Y1-H132A, and hB1-bradykinin receptors were not significantly different, suggesting that the presence of His at this position is essential for producing the wild-type hP2Y1 receptor effect on Zn2+ inhibition of the channel. A similar case can be made for hyperpolarization-induced activation, in that the hP2Y11 receptor exhibited properties that were significantly different from the wild-type hP2Y1 receptor but similar to the hB1-bradykinin receptor.

The results reported in this study and our previous work suggests that certain P2Y receptors are capable of modulating ion channel function through membrane-delimited interactions that do not appear to be associated with classical G-protein signaling mechanisms. The observation that mutations in TM3 produce significant changes in Tin channel function suggests that receptor TM domains may participate in channel regulation. Although a specific molecular interaction between the expressed P2Y receptor and the Tin channel has yet to be identified, one possible interpretation of our results might be a physical coupling between the two proteins. This would explain how relatively subtle changes in TM3 structure could produce changes within the Tin channel that lead to altered activation and Zn2+ sensitivity. Future experiments will be necessary to determine the molecular basis of Tin channel modulation by P2Y1 receptors.