On the role of endogenous G-protein beta gamma subunits in N-type Ca2+ current inhibition by neurotransmitters in rat sympathetic neurones

Abstract

1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.

Figure 1. The C-terminus of βARK1 reduces I_Ca inhibition by noradrenaline and somatostatin but not by angiotensin II

Superimposed Ca²⁺ current traces recorded in the absence or presence of noradrenaline (NA; 10 μm, A), somatostatin (Sst; 500 nm, B) and angiotensin II (Angio-II; 500 nm, C) in uninjected neurones (left panels, Control cells) and in neurones preinjected with 200 μg ml⁻¹ of the βARK1 construct (right panels). Ca²⁺ currents were elicited by the double-pulse protocol as illustrated in the inset. The outward currents elicited by the conditioning voltage pulse to +90 mV (at break) are omitted. In this and subsequent figures we have referred to the test depolarizations before and after the conditioning voltage pulse as ‘prepulse’ and ‘postpulse’, respectively. Responses to angiotensin II in C were recorded using the perforated-patch method where access resistances were 10.5 MΩ (left traces) and 8 MΩ (right traces). I_Ca inhibition was measured at steady state, 4 s after application of noradrenaline or somatostatin and 15–18 s after application of angiotensin II. Cells were recorded 48 h after injection. In this and subsequent figures, the horizontal dotted lines at the top of the traces indicate the zero current level.

Figure 3. βARK1 peptide expression prevents voltage-dependent inhibition of I_Ca

A, bar graph summarizing the effects of βARK1 peptide expression on I_Ca inhibition induced by noradrenaline (10 μm, filled bars), somatostatin (500 nm, shaded bars) and angiotensin II (500 nm, open bars). Uninj, uninjected control cells. Bars represent means ±s.e.m. for the number of cells indicated. Somatostatin and angiotensin II inhibition was recorded in cells preinjected with 200 μg ml⁻¹βARK1 construct. Cells were recorded 48 h after injection. *** P < 0.0001, compared with the respective controls. Inset, I_Ca amplitude was measured isochronally 4 ms after the onset of the test pulse (dashed line) from the zero current level obtained in the presence of cadmium (Cd²⁺, 500 μm). I_Ca was elicited from −70 to +5 mV. B, bar graph summarizing the effects of the βARK1 peptide on the facilitation ratio (postpulse : prepulse) in the absence or presence of neurotransmitters as indicated. □, control; ▪, βARK1 peptide. *** P < 0.0001.

Figure 2. SCG neurone preinjected with the βARK1 construct (100 μg ml⁻¹) displays strong immunoreactivity for the C-terminus of βARK1

Upper panel, fluorescence image of an intranuclearly injected neurone. Lower panel, immunostaining for the C-terminus domain of βARK1 48 h after injection. The star indicates the injected cell shown in the corresponding fluorescence image. Scale bar, 20 μm.

Figure 4. Modulation of I_Ca by GTP-γ-S in neurones overexpressing βARK1 peptide

Changes in facilitation (upper panel) and peak Ca²⁺ currents (lower panel) during dialysis of GTP-γ-S (500 μm) in an uninjected neurone (○), in an uninjected neurone incubated with PTX (1 μg ml⁻¹ for 24 h; ▿) and in a neurone preinjected with 400 μg ml⁻¹βARK1 construct (•). Time 0 refers to the time at which an adequate access resistance was obtained. I_Ca was elicited by voltage steps from −70 to +5 mV as in Fig. 3.

Figure 5. β₁γ₂ overexpression specifically occludes PTX-sensitive inhibition

A and B, prepulse I_Ca amplitude (○) and facilitation (♦) plotted as a function of time in a control neurone (A; preinjected with 800 μg ml⁻¹γ₂ construct, 31 pF) and in a neurone co-expressing β₁ and γ₂ subunits (B; 400 μg ml⁻¹ each of β₁ and γ₂ constructs, denoted β₁γ₂, 40 pF). Horizontal bars indicate the time and duration of application of somatostatin (50 nm), noradrenaline (300 nm) and angiotensin II (500 nm). The lower panel in B shows superimposed I_Ca traces selected before (open arrows) and during (filled arrows) application of agonists as indicated in the upper panel in B. I_Ca was recorded using the perforated-patch method and evoked using the double voltage pulse as illustrated in Fig. 1. Cells were recorded 24 h after injection.

Figure 6. Co-expression of the C-terminus domain of βARK1 prevents the effects of Gβ₁γ₂ dimers

A, perforated-patch clamp recordings of Ca²⁺ currents evoked using the double-pulse voltage protocol (as in Fig. 1) in a neurone preinjected with β₁- and γ₂-encoding plasmids (400 μg ml⁻¹, each; upper traces) and in a neurone in which the βARK1-generating plasmid (400 μg ml⁻¹; lower traces) was co-injected with the β₁ and γ₂ constructs. The dashed lines delineate the level of facilitated I_Ca following conditioning depolarization. B, bar graph summarizing the effects of β₁γ₂ and β₁γ₂+βARK1 overexpression on basal I_Ca density (□) and facilitation (▪). Bars represent means ±s.e.m. for the number of cells indicated. ** P < 0.001. Cells were recorded using the perforated-patch method, 24 h after injection.

Figure 7. The βARK1 peptide modifies decay of facilitation

A, representative examples of the decay of facilitation in an uninjected neurone (○) and in a neurone preinjected with 150 μg ml⁻¹βARK1 construct (•) in the presence of 10 μm noradrenaline. Peak currents were normalized to the current measured with a 2.5 ms interval between the conditioning step to +90 mV and the test step to +5 mV. Smooth curves are exponential fits to the data. The inset shows the protocol used to measure the time course of inhibition and some superimposed current trace recordings of the βARK1-expressing cell. B, I_Ca amplitude plotted as a function of time in an uninjected neurone (left panel) and in a neurone expressing the βARK1 peptide (100 μg ml⁻¹, right panel). The horizontal filled bars indicate the application of 10 μm noradrenaline and the dashed lines the time and duration for which iterative (4 Hz) depolarizations to +90 mV were applied. I_Ca was activated every 0.5–1 s, except immediately after the +90 mV conditioning depolarization (dashed line) where it was activated 10 ms, 80 ms, 200 ms and 1 s after the offset of the depolarization. Ca²⁺ currents were recorded in the whole-cell mode and elicited by voltage steps from −70 to +5 mV. C, superimposed current traces selected at the times indicated in B.