Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca(2+)-independent protein kinase C

Abstract

1. Members of the Kir3.0 family of inwardly rectifying K(+) channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by G(betagamma) subunits released in response to G(i/o)-coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the G(q/11)-coupled M(1) and M(3) muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal-type channels stably expressed in the human embryonic kidney cell line HEK293. 2. We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M(1) and M(3) receptors. This contrasts with the simple stimulation seen through activation of M(2) and M(4) receptors. 3. Channel stimulation via M(1) but not M(3) receptors was sensitive to pertussis toxin whereas channel inhibition through both M(1) and M(3) receptors was insensitive. In contrast over-expression of the C-terminus of phospholipase Cbeta1 or a G(q/11)-specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4. The inhibitory effects of M(1) and M(3) receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4alphaphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M(3) receptor. 5. The receptor-mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0-669 nM). 6. The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCdelta and PKCepsilon. 7. We also demonstrate that activation of such a pathway via both receptor-mediated and receptor-independent means profoundly attenuated subsequent channel stimulation by G(i/o)-coupled receptors. 8. Our data support a role for a Ca(2+)-independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M(1) and M(3) muscarinic receptor stimulation.

Figure 8. Expression and translocation of PKC isoforms in HEK293 cells

A, immunoblot illustrating endogenous expression of PKC isoforms in HEK293 cells. Molecular mass (in kDa) is indicated on the left. B, the translocation of PKCδ and PKCε was investigated in the HKIR3.1/3.2 cell line in response to PMA and the inactive analogue 4αphorbol (4α; both 100 nm). The immunoblots, which were quantified by gel-scanning densitometry (see Methods), are shown on the left-hand side of this figure and the corresponding bar charts on the right-hand side. C, cytosol; P, particulate. Data are presented as means ±s.d. (n = 3).

Figure 1. M₂ and M₄ muscarinic receptors stimulate Kir3.1+3.2A channels

A, a representative example of the effects of M₂ receptor stimulation (10 μm carbachol, CCh) on Kir3.1+3.2A currents. Currents were elicited by holding cells at 0 mV and stepping to potentials between −100 and +50 mV in 10 mV increments for 100 ms. Current traces were recorded before (Control), during (+carbachol) and after receptor stimulation (Wash). B, corresponding current–voltage relationships from data shown in A. C, an example of the effects of 10 μm carbachol (applied as indicated by the bar) upon membrane current in a cell voltage clamped at −60 mV. The dotted line indicates zero current and the dashed line indicates basal current prior to receptor stimulation. Note that after removal of carbachol current returns to basal level. D, bar charts summarizing the effects of stimulating M₂ and M₄ receptors in control (left-hand panel) and PTx-treated cells (right-hand panel). □, basal current density (Control); ▪, current density due to receptor stimulation (+CCh); , current density after agonist is removed (Wash). Numbers in parentheses indicate the number of cells recorded from. In control cells M₂ stimulation increased basal current density from 61.4 ± 8.4 to 214.9 ± 31.3 pA pF⁻¹(n = 16, **P < 0.01) whilst M₄ receptor stimulation increased current density from 82.9 ± 19.2 to 251.9 ± 47.6 pA pF⁻¹(n = 15, **P < 0.01). In PTx-treated cells (100 ng ml⁻¹, 16 h) carbachol was unable to potentiate Kir3.1+3.2A currents (M₂: basal 83.8 ± 15.1 pA pF⁻¹, +carbachol 95.0 ± 12.1 pA pF⁻¹, n = 6, P = 0.12; M₄: basal 53.3 ± 11.7 pA pF⁻¹, +carbachol 56.6 ± 13.8 pA pF⁻¹, n = 8, P = 0.44).

Figure 2. M₁ and M₃ muscarinic receptors have a dual effect on Kir3.1+3.2A channels

A, example of currents (elicited as described in Fig. 1A) recorded from HKIR3.1/3.2 cells co-expressing M₁ receptors. Currents were recorded before (Control), during (+carbachol) and 5 min after receptor stimulation (Wash). B, current-voltage relationships from the data shown in A. C, representative examples of the effects of M₁ and M₃ receptor stimulation on Kir3.1+3.2A current recorded at a holding potential of −60 mV. Carbachol (10 μm) was applied as indicated by the bar, the dotted line indicates zero current and the dashed line indicates basal current prior to receptor stimulation. The left-hand panel shows the effects of M₁ receptor stimulation, the middle panel the effects of M₃ receptor stimulation and the right-hand panel the effects of co-expression of β₁γ₂ dimers upon the M₃ response. D, bar charts summarizing the effects of stimulating M₁, M₃ and M₃+β₁γ₂ on Kir3.1+3.2A currents (M₁: basal 60.0 ± 13 pA pF⁻¹, +carbachol 123.7 ± 14.0 pA pF⁻¹, wash 24.1 ± 7.6 pA pF⁻¹, equivalent to 62.1 ± 4.8 % inhibition (n = 23); M₃: basal 28.1 ± 4.9 pA pF⁻¹, +carbachol 89.2 ± 9.2 pA pF⁻¹, wash 13.2 ± 2.3 pA pF⁻¹, equivalent to 47.6 ± 4.4 % inhibition (n = 31); M₃+β₁γ₂: basal 136.8 ± 28.1 pA pF⁻¹, +carbachol 159.9 ± 24.9 pA pF⁻¹, wash 68.9 ± 20 pA pF⁻¹, equivalent to 53.2 ± 6.6 % inhibition (n = 12)). **P < 0.01; ***P < 0.001.

Figure 3. The period of application of carbachol does not affect the extent of inhibition of Kir3.1+3.2A currents

A, representative traces illustrating the effect of different periods of application (2, 20 and 200 s) of carbachol (10 μm) on HKIR3.1/3.2/M3 cells voltage clamped at −60 mV. The dotted line indicates basal current prior to receptor stimulation. B, bar charts summarizing data from a number of cells with the periods of carbachol application as indicated in the corresponding data traces shown in A.□, basal current density prior to carbachol application (Control); ▪, peak current density during application (+CCh peak); , current density measured at the end of the drug application (+CCh end); , current density after the removal of stimulus (Wash). Levels of significance are shown with respect to Control current density. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. The inhibitory effect of M₃ receptor stimulation on Kir3.1+3.2A channels is Ca²⁺ independent

This bar chart summarizes the percentage inhibition of Kir3.1+3.2A current density mediated by M₃ receptor stimulation (10 μm CCh) using four different concentrations of calculated intracellular free Ca²⁺ (0, 18, 111 and 669 nm). These experiments were performed in the stable cell line HKIR3.1/3.2/M3. *P < 0.05.

Figure 5. Inhibition of Kir3.1+3.2A channels by M₁ and M₃ receptors is not mediated through G_i/o proteins

A, current traces recorded from a PTx-treated cell before (Control), during (+carbachol) and after M₁ receptor stimulation (Wash). B, current-voltage relationships obtained from the data shown in A. C, bar charts summarizing the effects of PTx upon M₁ (left-hand panel) and M₃ (right-hand panel) responses. PTx abolishes M₁-mediated channel activation (▪) but not inhibition ), but is ineffective on both channel activation and inhibition due to M₃ receptor stimulation. D, the C-terminus of PLCβ1 (PLCβ1ct) attenuated M₃-mediated inhibition (M₃+PLCβ1ct: basal 13.9 ± 3.2 pA pF⁻¹, +carbachol 55.7 ± 15.3 pA pF⁻¹, wash 11.6 ± 4.2 pA pF⁻¹(n = 9)). Thus M₃-mediated percentage inhibition was reduced from 78.7 ± 2.7 %(n = 16) in the absence of PLCβ1ct to 23.5 ± 10.6 %(n = 9, P < 0.001) in its presence. These experiments were performed in the HKIR3.1/3.2/M3 stable cell line. E, bar chart summarizing the effects of co-expression of RGS2 upon M₁ and M₃ responses (M₁+RGS2: basal 22.3 ± 3.8 pA pF⁻¹, +carbachol 74.0 ± 21.3 pA pF⁻¹, wash 15.0 ± 2.5 pA pF⁻¹(n = 5); M₃+RGS2: basal 47.7 ± 10.0 pA pF⁻¹, +carbachol 110.3 ± 36.3 pA pF⁻¹, wash 38.9 ± 10.0 pA pF⁻¹(n = 8)). RGS2 reduced M₁-mediated percentage inhibition from 62.1 ± 4.8 %(n = 23) to 29.6 ± 8.7 %(n = 5, P < 0.01) and M₃-mediated percentage inhibition from 47.6 ± 4.4 %(n = 31) to 20.0 ± 11.7 %(n = 8, P = 0.01). *P < 0.05; **P < 0.01.

Figure 6. M₃-mediated channel inhibition involves a Ca²⁺-independent PKC isozyme

A, the effects of staurosporine (1 μm), GF109203X (3 μm) and Ro-31-8220 (3 μm) on M₃-mediated channel inhibition. PKC inhibitors were applied for at least 5 min prior to M₃ receptor stimulation with 10 μm carbachol and the percentage inhibition of basal current density measured. □, percentage inhibition of Kir3.1+3.2A currents by M₃ receptor stimulation; ▪, percentage inhibition in the presence of PKC inhibitor. Experiments using the inhibitor GF109203X were performed in HKIR3.1/3.2 cells transiently transfected with M₃ receptors whilst the experiments using staurosporine and Ro-31-8220 were done in the stable cell line HKIR3.1/3.2/M3. B, illustration of the effects of DOG (5 μm) on basal Kir3.1+3.2A currents over a 3 min time period. Current traces are shown at 1 min intervals (time points, in minutes, are indicated above each trace) during the 3 min application of DOG and after 5 min wash. C, bar chart summarizing the inhibitory effects of PMA, PDBu (both 100 nm), DOG (5 μm) and 4αphorbol (100 nm) on Kir3.1+3.2A basal current density. For comparison the effects of carbachol on the M₃ receptor are also shown. We compared current density before and after application of PDBu, PMA, DOG or 4αphorbol using Student's paired t test. The PKC activators PMA, PDBu and DOG all significantly reduced current density (pre-PDBu: 64.9 ± 15.7 pA pF⁻¹, post-PDBu: 28.3 ± 10.5 pA pF⁻¹, n = 6, P < 0.01; pre-PMA: 32.5 ± 8.6 pA pF⁻¹, post-PMA: 13.7 ± 4.3 pA pF⁻¹, n = 6, P = 0.01; pre-DOG: 35.4 ± 5.2 pA pF⁻¹, post-DOG: 4.7 ± 0.9 pA pF⁻¹, n = 11, P < 0.001). In contrast 4αphorbol had no significant effects upon current density (pre-4αphorbol: 23.72 ± 6.61 pA pF⁻¹, post-4αphorbol: 20.8 ± 3.07 pA pF⁻¹, n = 6, P = 0.7). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. Occlusion of M₃ receptor-mediated responses by prior stimulation with DOG

A, current-voltage traces elicited as described in Fig. 1A were recorded in HKIR3.1/3.2/M3 cells. The left-hand panel illustrates a control trace. DOG (5 μm) was then applied until its effects reached a maximum and another trace recorded (centre panel). Carbachol (10 μm) was then applied (in the presence of DOG) for 20 s during which currents were increased (B). After the removal of carbachol another current-voltage trace was recorded (right-hand panel). B, trace recorded at −60 mV from the same cell as illustrated in A. The lower-case letters a-c refer to where data were measured for the bar chart shown in C. C, bar chart illustrating the effects of carbachol on current density when applied after prior inhibition by DOG. Data are normalized to the control current prior to the application of DOG. *P < 0.05.

Figure 9. The effects of M₁ or M₃ receptor stimulation prevent subsequent channel activation by the G_i/o-coupled A₁ receptor

A, individual current traces recorded from a HKIR3.1/3.2/M3 cell transiently transfected with the A₁ receptor. The A₁ receptor was stimulated with 1 μm NECA prior to and following M₃ stimulation with 10 μm carbachol. Time points (in seconds) are indicated above each trace (the experiment was started at t = 0). B, bar chart summarizing the effects of M₃ receptor stimulation on A₁-induced channel activation recorded in HKIR3.1/3.2/M3 cells transiently transfected with A₁ receptors. NECA-induced currents were measured at −60 mV before (95.9 ± 26.2 pA pF⁻¹) and after M₃ stimulation (32.8 ± 8.7 pA pF⁻¹, n = 5, P = 0.02). C, bar chart summarizing the effects of M₁ and M₃ receptor stimulation and DOG and PMA treatment upon NECA-induced Kir3.1+3.2A currents measured in the HKIR3.1/3.2/A1 line. Control NECA-induced currents: 125.9 ± 16.9 pA pF⁻¹, n = 31; NECA-induced currents after M₁ stimulation: 38.5 ± 10.0 pA pF⁻¹, n = 13, P < 0.01; NECA-induced currents after M₃ stimulation: 17.7 ± 4.8 pA pF⁻¹, n = 12, P < 0.001; NECA-induced currents following DOG: 22.4 ± 6.3 pA pF⁻¹, n = 11, P < 0.001; NECA-induced currents following PMA: 33.15 ± 13.22 pA pF⁻¹, n = 7, P = 0.02. *P < 0.05; **P < 0.01; ***P < 0.001.