The role of constitutive PKA-mediated phosphorylation in the regulation of basal I(Ca) in isolated rat cardiac myocytes

Abstract

1. Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca(2+) current (I(Ca)) observed in the absence of exogenous beta-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I(Ca) in the perforated patch-clamp configuration. 2. Calyculin A and isoprenaline (both 1 micromol l(-1)) increased basal I(Ca) (P<0.05), whereas H-89 inhibited I(Ca) in a concentration-dependent manner with an IC(50) approximately 5 micromol l(-1). H-89 also inhibited the response to 1.0 micromol l(-1) isoprenaline, although relatively high concentrations (30 micromol l(-1)) were required to achieve complete suppression of the response. 3. Double-pulse protocols were used to study the effects of 10 micromol l(-1) H-89 on time-dependent recovery of I(Ca) from voltage-dependent inactivation as well as the steady-state gating of I(Ca). T(0.5) (time for I(Ca) to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4. Steady-state activation/inactivation properties of I(Ca) were unaffected by 10 micromol l(-1) H-89 or 1 micromol l(-1) calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V(0.5) for activation and inactivation became more negative. 5. Data show that basal I(Ca) is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied beta-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to beta-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

Figure 1

Effects of calyculin A and isoprenaline (both 1 μmol l⁻¹) on I_Ca. (a) Representative tracings of I_Ca elicited by square depolarising pulses from −40 to 0 mV in the absence and presence of calyculin A and isoprenaline. (b) I–V curves for I_Ca obtained in response to 5 mV step increments from −40 to 60 mV. Each data point is the mean±s.e.m. of 8–10 different cells. Standard errors not visible are smaller than the symbols.

Figure 2

Effects of H-89 on I_Ca in isolated rat ventricular myocytes. (a) Representative original traces of I_Ca at differing concentrations of H-89. (b) I–V curves of I_Ca in the absence and presence of different concentrations of H-89. (c) Concentration–effect curve for H-89 at 35 and 25°C. Each data point is the mean±s.e.m. from six to eight cells.

Figure 3

Effects of H-89 on the response to isoprenaline. The response to isoprenaline was determined following equilibration of myocytes with either 10 (a and b) or 30 μmol l⁻¹ (c and d) H-89. The number above each bar is the number of separate myocytes tested. All the bars shown were significantly different from one another, except the effects of 30 μmol l⁻¹ H-89 vs 30 μmol l⁻¹ H-89 plus isoprenaline.

Figure 4

Effects of H-89, calyculin A and isoprenaline on recovery of I_Ca from voltage-dependent inactivation. (a) The inset in the bottom part of the figure shows the double-pulse protocol during which myocytes were depolarised from −40 to 0 mV with a progressively increasing interpulse interval (20 ms increments). The other panels in (a) show representative tracings illustrating the recovery of I_Ca in control conditions and in the presence of calyculin A (1 μmol l⁻¹), isoprenaline (1 μmol l⁻¹) and H-89 (10 μmol l⁻¹). (b) Mean (±s.e.m.) time course of I_Ca recovery fitted with the Boltzmann equation. (c) Mean (±s.e.m.) T_0.5 under control conditions (n=6) and in the presence of calyculin A (n=6), isoprenaline (n=4) and H-89 (n=9). ^*P<0.05.

Figure 5

Effects of cayculin A, isoprenaline and H-89 on voltage-dependent activation and inactivation of I_Ca. Myocytes were stimulated using a double-pulse protocol where the voltage of the first pulse was increased in 5 mV step increments (400 ms) from −40 to 60 mV followed by a second step to 0 mV after 2 ms. I_Ca recorded were converted to conductance (see text). (a) I_Ca during the second pulse to 0 mV in control conditions and in the presence of 1 μmol l⁻¹ calyculin A, 1 μmol l⁻¹ isoprenaline and 10 μmol l⁻¹ H-89. In each case, the largest amplitude I_Ca was recorded during the first step in the protocol. (b) Steady-state inactivation curves. (c) Steady-state activation curves. (d) Mean (±s.e.m.) voltage at half-maximal conductance for inactivation. (e) Mean (±s.e.m.) voltage for half-maximal conductance for activation. Both activation and inactivation curves were fitted with the Boltzmann function. Mean data are from 6–8 separate myocytes. ^*P<0.05.