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Ectodomain lysines and suramin block of P2X1 receptors.

Sim JA, Broomhead HE, North RA - J. Biol. Chem. (2008)

Bottom Line: ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors.The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449.The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom. joan.sim@manchester.ac.uk

ABSTRACT
P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

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Introduction of lysine residues into the mouse receptor can increase sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type mouse P2X1 receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the increased effectiveness of NF449 (3 nm) as an antagonist in the case of mouse P2X1[E138K] and of mouse P2X1[4K] receptors.
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fig3: Introduction of lysine residues into the mouse receptor can increase sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type mouse P2X1 receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the increased effectiveness of NF449 (3 nm) as an antagonist in the case of mouse P2X1[E138K] and of mouse P2X1[4K] receptors.

Mentions: Whole Cell Recording and Application of Agonists and Antagonists—All of the cells were pretreated with apyrase (2 units/ml, Type VII; Sigma) for at least 2 h before the recordings were commenced. The coverslips with attached cells were placed in a recording chamber mounted on the stage of an Axiovert microscope (Carl Zeiss). Extracellular recording solution containing 147 mm NaCl, 3 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, and 13 mm d-glucose, 13 (pH adjusted to 7.4 with NaOH) was superfused at a rate of 5.5 ml/min. Whole cell recordings were made at room temperature (20–23 °C) using an EPC9 amplifier, and data were collected using Pulse software (HEKA). The membrane potential was held at –60 mV. Patch electrodes and “puffer” electrodes (for ATP application) were pulled from glass pipettes (Harvard Apparatus, Edenbridge, UK) on a vertical puller (HEKA) and ranged from 6 to 9 mΩ in resistance when filled with an intracellular solution containing 147 mm NaCl, 10 mm HEPES, 10 mm EGTA (pH adjusted to 7.3 with NaOH). ATP solutions were prepared on the day of recording by diluting 100 mm frozen stock solution (pH adjusted to 7.3) in the external recording solution. Suramin and NF449 were prepared as 10 mm frozen stock and diluted to the required concentration on the day of recording currents. ATP was applied via a glass puffer pipette (1-μm tip diameter, ≈10 p.s.i., 69 kPa) using a pneumatic PicoPump (PV830; World Precision Instruments). The tip of the puffer pipette was positioned downstream from the cell with respect to the direction of flow of the superfusing solution and temporarily repositioned to a point ∼15 μm from the cell only for the period of application. With a concentration of ATP >1 μm, second applications 2 min after the first evoked a current that was less than 20% of the first response. Therefore, all concentration-response relations shown in this study were constructed from pooled data, in which ATP (0.001–100 μm) was applied only once to each cell on each coverslip. Accordingly, the same data points for control concentration-response curves appear more than once in the panels of Figs. 2, 3, 4, 5. Suramin and NF449 were applied in the superfusing solution for 5–10 min prior to the application from the puffer pipette of a solution containing both ATP and the appropriate antagonist. The currents evoked were then compared with those observed in other cells with no antagonist pretreatment.


Ectodomain lysines and suramin block of P2X1 receptors.

Sim JA, Broomhead HE, North RA - J. Biol. Chem. (2008)

Introduction of lysine residues into the mouse receptor can increase sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type mouse P2X1 receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the increased effectiveness of NF449 (3 nm) as an antagonist in the case of mouse P2X1[E138K] and of mouse P2X1[4K] receptors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2573084&req=5

fig3: Introduction of lysine residues into the mouse receptor can increase sensitivity to NF449. A, representative traces of membrane currents evoked by ATP (10 μm), applied for 1 s as indicated by the horizontal bars. Left panels, control. Center and right panels, in NF449 (3 and 300 nm). B, ATP concentration-response curves for wild type mouse P2X1 receptors and for receptors with one or four lysines introduced at the positions indicated. Open symbols, control. Solid symbols, in presence of NF449 (3 and 300 nm). Note the increased effectiveness of NF449 (3 nm) as an antagonist in the case of mouse P2X1[E138K] and of mouse P2X1[4K] receptors.
Mentions: Whole Cell Recording and Application of Agonists and Antagonists—All of the cells were pretreated with apyrase (2 units/ml, Type VII; Sigma) for at least 2 h before the recordings were commenced. The coverslips with attached cells were placed in a recording chamber mounted on the stage of an Axiovert microscope (Carl Zeiss). Extracellular recording solution containing 147 mm NaCl, 3 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, and 13 mm d-glucose, 13 (pH adjusted to 7.4 with NaOH) was superfused at a rate of 5.5 ml/min. Whole cell recordings were made at room temperature (20–23 °C) using an EPC9 amplifier, and data were collected using Pulse software (HEKA). The membrane potential was held at –60 mV. Patch electrodes and “puffer” electrodes (for ATP application) were pulled from glass pipettes (Harvard Apparatus, Edenbridge, UK) on a vertical puller (HEKA) and ranged from 6 to 9 mΩ in resistance when filled with an intracellular solution containing 147 mm NaCl, 10 mm HEPES, 10 mm EGTA (pH adjusted to 7.3 with NaOH). ATP solutions were prepared on the day of recording by diluting 100 mm frozen stock solution (pH adjusted to 7.3) in the external recording solution. Suramin and NF449 were prepared as 10 mm frozen stock and diluted to the required concentration on the day of recording currents. ATP was applied via a glass puffer pipette (1-μm tip diameter, ≈10 p.s.i., 69 kPa) using a pneumatic PicoPump (PV830; World Precision Instruments). The tip of the puffer pipette was positioned downstream from the cell with respect to the direction of flow of the superfusing solution and temporarily repositioned to a point ∼15 μm from the cell only for the period of application. With a concentration of ATP >1 μm, second applications 2 min after the first evoked a current that was less than 20% of the first response. Therefore, all concentration-response relations shown in this study were constructed from pooled data, in which ATP (0.001–100 μm) was applied only once to each cell on each coverslip. Accordingly, the same data points for control concentration-response curves appear more than once in the panels of Figs. 2, 3, 4, 5. Suramin and NF449 were applied in the superfusing solution for 5–10 min prior to the application from the puffer pipette of a solution containing both ATP and the appropriate antagonist. The currents evoked were then compared with those observed in other cells with no antagonist pretreatment.

Bottom Line: ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors.The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449.The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom. joan.sim@manchester.ac.uk

ABSTRACT
P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

Show MeSH
Related in: MedlinePlus