Regulation of the muscarinic K+ channel by extracellular ATP through membrane phosphatidylinositol 4,5-bisphosphate in guinea-pig atrial myocytes

Abstract

1 The present study was designed to examine the functional role of membrane phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the regulation of the muscarinic K(+) channel (I(K,ACh)) by extracellular ATP and adenosine in guinea-pig atrial myocytes, using the whole-cell patch-clamp method. 2 Bath application of ATP in micromolar concentrations typically evoked a transient activation of I(K,ACh); a rapid activation phase was consistently followed by a progressive decline even to the baseline level despite the continued presence of ATP. This progressive decline of I(K,ACh) was significantly attenuated either by blockade of phospholipase C (PLC) with compound 48/80 (100 microM) or by addition of PtdIns(4,5)P(2) (50 microM) to the cell inside, suggesting that depletion of membrane PtdIns(4,5)P(2) via PLC activation is mainly, if not totally, responsible for the progressive decline of I(K,ACh) during the presence of ATP. 3 When atrial myocytes were exposed to wortmannin (50 microM) following ATP (50 microM) application to impair the resynthesis of PtdIns(4,5)P(2), the activation of I(K,ACh) evoked by subsequently applied ATP (50 microM) was greatly reduced. Activation of I(K,ACh) by adenosine (100 microM) was partially reduced by pretreatment of atrial myocytes with ATP (100 microM) and was largely abolished by a further addition of wortmannin (50 microM) in the presence of ATP (100 microM). These results support the view that the activation of I(K,ACh) by ATP and adenosine depends on membrane PtdIns(4,5)P(2) that is subject to reduction by extracellular ATP. 4 The present study thus provides functional evidence to suggest that extracellular ATP activates PLC and thereby depletes membrane PtdIns(4,5)P(2) that is critically involved in the activation process of I(K,ACh) by its agonists ATP and adenosine in guinea-pig atrial myocytes.

Figure 1

Effect of bath application of ATP on whole-cell currents in guinea-pig atrial myocytes. (a) Chart record of whole-cell current (Im) recorded at a holding potential of −40 mV and during voltage ramps between +50 and −130 mV at 0.4 V s⁻¹ (see Methods). Rapid deflections represent changes in membrane current in response to the voltage ramps applied before (1), and during exposure to 50 μM ATP (2 and 3), and after washout of the ATP (4). (b) Superimposed I–V relationships obtained using the voltage ramps applied at the time points indicated by numerals (1–4) in (a). (c) Superimposed I–V relationships for the difference currents obtained by digital subtraction of current records as indicated.

Figure 2

Effect of PLC inhibition or PtdIns(4,5)P₂ addition on the progressive decline of I_K,ACh during exposure to ATP. (a, c) Time course of changes in the holding current level at −40 mV during ∼2.5 min exposure to ATP at concentrations of 10 μM (a) and 50 μM (c) in atrial myocyte dialyzed with a pipette solution containing either 100 μM compound 48/80 (a) or 50 μM PtdIns(4,5)P₂ (c). The myocyte was stimulated with extracellular ATP 15–20 min after rupture of the patch membrane (establishment of whole-cell mode) with each reagent. (b, d) Percentage decrease of I_K,ACh during exposure to ATP at 10 μM (b) and 50 μM (d) in myocytes dialyzed with control pipette solution (Control) and pipette solution supplemented with either 100 μM compound 48/80 (b) or 50 μM PtdIns(4,5)P₂ (d). Percentage decrease was determined at the current levels measured at the end of ∼2.5 min exposure to ATP. Inset in (a) shows a representative time course of I_K,ACh (at −40 mV) during exposure to 10 μM ATP, recorded from an atrial myocyte dialyzed with a control pipette solution. ^*P<0.05 and ^**P<0.01 compared with control value (Student's unpaired t-test).

Figure 3

Effect of wortmannin on the activation of I_K,ACh by extracellular ATP. (a) An atrial myocyte was stimulated with twin-pulse applications of 50 μM ATP with an interval of ∼4 min, as indicated by horizontal bars. (b, c) Following the first stimulation with 50 μM ATP, the atrial myocyte was then exposed to wortmannin at a concentration of 50 nM (b) or 50 μM (c) for ∼4 min and was again stimulated with 50 μM ATP, as indicated. (d) Summary data for current ratio of I_P2/I_P1, obtained by normalizing the peak amplitude of I_K,ACh during the second application of ATP (I_P2) with reference to that during the first ATP application (I_P1), expressed as percentage. Asterisks represent P-values according to Tukey's multiple means comparison test (^**P<0.01). There was no significant difference between the control and wortmannin (50 nM) groups, but the difference was significant (P<0.01) when the wortmannin (50 μM) group was compared with either the control or wortmannin (50 nM) group.

Figure 4

Effect of pretreatment with wortmannin and/or ATP on adenosine-evoked I_K,ACh. (a) Adenosine (100 μM) was twice added to the same myocyte with an interval of ∼6 min, which elicited I_K,ACh of almost similar magnitude. (b) Following the first stimulation with 100 μM adenosine, an atrial myocyte was exposed to 50 μM wortmannin for ∼4 min and subsequently stimulated with 100 μM adenosine. (c–e) The atrial myocytes were exposed to 100 μM ATP (c), 100 μM ATP plus 50 nM wortmannin (d) or 100 μM ATP plus 50 μM wortmannin (e) for ∼4 min before the second application of 100 μM Ado. (f) Current ratio of I_P2/I_P1, obtained by normalizing the peak amplitude of I_K,ACh during the second application of 100 μM adenosine (I_P2) with reference to that during the first adenosine application (I_P1), from the experimental protocols shown in (a–e). Asterisks represent P-values according to Tukey's multiple means comparison test (^**P<0.01). Note that there was no significant difference in the current ratio (I_P2/I_P1) between the ATP alone and the ATP plus wortmannin (50 nM) application groups, but the difference was highly significant (P<0.01) when the ATP plus wortmannin (50 μM) application group was compared with either the ATP alone or ATP plus wortmannin (50 nM) application group (f).

Figure 5

Inhibition of adenosine-evoked I_K,ACh by extracellular ATP. (a) Chart records of whole-cell currents (Im) recorded at a holding potential of −40 mV and during voltage ramps applied before (1), and during exposure to 100 μM adenosine (2), and after further addition of 100 μM ATP (3 and 4). The rapid deflections in the current recording reflect the imposition of voltage-ramp protocols. (b) Superimposed I–V relationships measured during the voltage ramps applied at the points indicated by numerals (1–4) in (a). (c) Superimposed I–V relationships for the difference currents obtained by digital subtraction of current records as indicated.