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Purinergic Signal
2004 Dec 01;11:75-81. doi: 10.1007/s11302-004-4744-5.
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P2Y(1) receptor modulation of endogenous ion channel function in Xenopus oocytes: Involvement of transmembrane domains.
Lee SY
,
Nicholas RA
,
O'Grady SM
.
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Agonist activation of the hP2Y(1) receptor expressed in Xenopus oocytes stimulated an endogenous voltage-gated ion channel, previously identified as the transient inward (T(in)) channel. When human P2Y(1) (hP2Y(1)) and skate P2Y (sP2Y) receptors were expressed in Xenopus oocytes, time-to-peak values (a measure of the response to membrane hyperpolarization) of the T(in) channel were significantly reduced compared to oocytes expressing the hB(1)-bradykinin receptor or the rat M(1)-muscarinic (rM(1)) receptor. Differences in activation were also observed in the T(in) currents elicited by various P2Y receptor subtypes. The time-to-peak values of the T(in) channel in oocytes expressing the hP2Y(4), hP2Y(11), or hB(1)-bradykinin receptors were similar, whereas the channel had significantly shorter time-to-peak values in oocytes expressing either the hP2Y(1) or sP2Y receptor. Amino acid substitutions at His-132, located in the third transmembrane domain (TM3) of the hP2Y(1) receptor, delayed the onset of channel opening, but not the kinetics of the activation process. In addition, Zn(2+) sensitivity was also dependent on the subtype of P2Y receptor expressed. Replacement of His-132 in the hP2Y(1) receptor with either Ala or Phe increased Zn(2+) sensitivity of the T(in) current. In contrast, truncation of the C-terminal region of the hP2Y(1) receptor had no affect on activation or Zn(2+) sensitivity of the T(in) channel. These results suggested that TM3 in the hP2Y(1) receptor was involved in modulating ion channel function and blocker pharmacology of the T(in) channel.
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.
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.
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 V50 values and slope factors for each conductance are listed in Table 1.
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.
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.
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