The role of ryanodine receptors in the cyclic ADP ribose modulation of the M-like current in rodent m1 muscarinic receptor-transformed NG108-15 cells

Abstract

1. The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M-like current (I_K(M,ng)) by acetylcholine was investigated in m1 muscarinic receptor-transformed mouse neuroblastoma-rat glioma hybrid (NG108-15) cells using patch-clamp techniques and calcium microfluorimetry.

2. Acetylcholine (1–100 μm) decreased I_K(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8-amino-cyclic ADP ribose (10–100 μm) and 8-bromo-cyclic ADP ribose (100–1000 μm) reduced this inhibition of I_K(M,ng) in a concentration-dependent manner. The half-maximal inhibition concentrations for 8-amino- cyclic ADP ribose and 8-bromo-cyclic ADP ribose were around 40 μm and 1 mm, respectively.

3. Neither of the cyclic ADP ribose antagonists altered the amplitude of I_K(M,ng)per se, or the incidence of the concurrent Ca²⁺-activated K⁺ current (I_IK(Ca)) activation, also mediated by acetylcholine.

4. The ryanodine receptor modulators ryanodine (1–10 μm) and Ruthenium Red (10 μm) did not alter I_K(M,ng) amplitude or I_K(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red.

5. Intracellular calcium levels measured with fura-2 microfluorimetry were increased with low concentrations of ryanodine (1 μm), more consistently with caffeine (10 mm), and in almost every case with both bradykinin (300 nm) and acetylcholine (100 μm). Caffeine-, but not bradykinin-evoked responses were abolished by preincubation with ryanodine (10 μm).

6. The fast ‘rundown rate’ of the M-current recorded in rat superior cervical ganglion cells under whole-cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 μM) was applied directly to the cytoplasmic face of inside-out membrane patches excised from rat superior cervical ganglion cells containing M-channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening).

7. These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate I_K(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP ribose may not act directly on the M-channels in inside-out patches.

The nature of the intracellular link between receptor activation and M-current (I_K(M)) inhibition has been the subject of investigation for over 15 years and many of the results are still controversial. Amongst the few undisputed facts is that a pertussis toxin-insensitive G-protein is involved, which is likely to be one of the Gq family (Jones et al. 1995), and that a mobile and/or cytosolic diffusable messenger may be required (Selyanko et al. 1992, 1995; Marrion, 1996). The identity of this diffusable messenger is still under much debate, as is the molecular identity of the channel itself (see Marrion, 1997a). However, a consistency between the results over these last 15 years is the accumulated evidence that the inhibition is concomitant with changes in intracellular calcium levels (see Marrion, 1997b). This may mean either that calcium acts directly or in concert with another messenger or messengers to inhibit I_K(M) (Selyanko & Brown, 1996a), or that the transduction pathway activates a messenger that can simultaneously alter calcium levels and modulate M-channels.

Differentiated neuroblastoma × glioma hybrid (NG108-15) cells display a time- and voltage-dependent potassium current with kinetic and pharmacological characteristics very similar to the I_K(M) in rat superior cervical ganglion cells, which has been designated I_K(M,ng) (Robbins et al. 1992). Inhibition of I_K(M,ng) by G-protein-coupled, phospholipase C-linked receptors is a robust event in NG108-15 cells. In a previous study, a large number of putative mobile messengers were ruled out in neuroblastoma × glioma cells (Robbins et al. 1993). More recently, it has been reported that alterations in β-NAD⁺ levels (a precursor of cyclic adenosine diphosphate ribose, cADPR) by streptozotocin can modulate the inhibition of I_K(M,ng) by acetylcholine in m1 muscarinic receptor-transformed NG108-15 cells. Furthermore, intracellular application of cADPR can inhibit I_K(M,ng) in the absence of receptor activation (Higashida et al. 1995) and streptozotocin can alter bradykinin-, ATP-, angiotensin II- and endothelin 1-mediated inhibition of the I_K(M,ng) in a similar way (Higashida et al. 1996). c-ADPR has already been suggested as a calcium-mobilizing messenger through acting as an activator or co-activator of the ryanodine receptor in a number of cell types (see Galione & White, 1994). Indeed, it can increase intracellular calcium levels in NG108-15 cells (Higashida et al. 1995). This messenger could then account for the close coupling between inhibition of I_K(M) and the associated changes in intracellular calcium. A number of molecular targets for cADPR have been suggested. These include the ryanodine receptor protein itself (Sitsapesan et al. 1994; Clementi et al. 1996), calmodulin (Lee et al. 1994; Tanaka & Tashjian, 1995), FK506 binding protein (Noguchi et al. 1997) and one or more unidentified cADPR binding proteins (Walseth et al. 1993).

Here we report that the modulation of I_K(M,ng) inhibition by cyclic ADPR is a separate event from its calcium-mobilizing role, not involving the ryanodine receptor, but an as yet unidentified molecular target. A preliminary abstract of some of the work has been published (Robbins et al. 1995).

METHODS

Cell culture

Neuroblastoma × glioma cells

m1 muscarinic receptor-transformed neuroblastoma × glioma hybrid (NG108-15) cells (obtained from Professor H. Higashida, Kanazawa, Japan) were cultured as described previously (Robbins et al. 1993). Briefly, they were continuously grown at 37°C in Dulbecco's modified Eagle's medium (DMEM, high glucose) containing 5 % fetal calf serum (FCS; myoclone), 30 μM hypoxanthine, 1.2 μM aminopterine, 4.8 μM thymidine and 2 mM L-glutamine, in the presence of 10 % CO₂. Cells were grown in 25 ml flasks to 70–80 % confluence then passaged 1 : 3 every 3–4 days. For electrophysiological recordings, cells were transferred to 35 mm Petri dishes (density, 2000–5000 cells dish⁻¹) which had been precoated with polyornithine. For microfluorimetry, cells were plated onto polyornithine-coated coverslips (22 mm × 22 mm). Cells were differentiated 24 h after plating by replacing the growing medium with one in which the FCS had been reduced to 1 %, aminopterine had been omitted, and PGE₁ (10 μM) and 3-isobutyl-1-methylxanthine (IBMX; 50 μM) had been added.

Rat superior cervical ganglion cells

Sprague-Dawley rats (15–20 days old) were terminally anaesthetized by a rising concentration of CO₂ and decapitated. The superior cervical ganglion was isolated, freed from connective tissue and cut into four. The tissue was washed in Hank's Ca^{2 +}- and Mg^{2 +} -free balanced salt solution then dissociated in collagenase (800 i.u.) for 30 min at 37°C in 5 % CO₂ atmosphere. After further washes, the tissue was placed in a trypsin solution (bovine Type XIIS) for 30 min. The reaction was stopped by dilution in growth medium (see below), and cells were centrifuged at 190 g for 4 min, resuspended in growth medium and gently triturated with a glass pipette. The resultant suspension was diluted (1 : 3 in growth medium) and plated onto poly-l-lysine-coated 35 mm dishes (Nunc). Cells were used 1–2 days after plating. Until then, cells were maintained at 37°C (5 % CO₂) in growth medium of the following composition: L15 medium, 10 % FCS, 2 mM L-glutamine, 38 mM D-glucose, 24 mM sodium bicarbonate, 5750 i.u. penicillin, 5.75 mg streptomycin and 50 ng ml⁻¹ 7S nerve growth factor.

Electrophysiological recordings

Whole-cell currents

The whole-cell variant of the patch-clamp technique was employed to record membrane currents from NG108-15 cells at 4–14 days after differentiation, similar to the procedure described in Robbins et al. (1993). Cells were superfused, at 5–10 ml min⁻¹, with a solution containing (mM): NaCl, 120; KCl, 3; glucose, 11; NaHCO₃, 22.6; MgCl₂, 1.2; Hepes, 5; and CaCl₂, 2.5; with 500 nM tetrodotoxin. The pH was 7.36 when gassed with 95 % O₂ and 5 % CO₂ at 35°C. Glass microelectrodes were used with a resistance of 2–4 MΩ when filled with (mM): potassium acetate, 90; KCl, 20; MgCl₂, 3; Hepes, 40; EGTA, 3; CaCl₂, 1; ATP, 2 mM; and GTP, 0.5; pH adjusted to 7.4 with KOH (1 M). The calculated free calcium concentration for this solution was 66 nM (Chelator for Windows 1.1) and series resistance was typically 5 MΩ.

Cells were routinely clamped at −20 to −30 mV and hyperpolarized by −30 or −40 mV to generate deactivating tail currents. Voltage pulses were generated by an amplifier (Axoclamp-2B, Axon Instruments) in discontinuous mode, while currents were recorded via a signal conditioner (CyberAmp 320, Axon Instruments), digitized on an ADC (Digidata 1200, Axon Instruments) and displayed on a PC running commercial software (pCLAMP 6, Axon Instruments). All whole-cell data were stored on an optical disk (Panasonic, UK).

Single channel recordings

M-channel activity was recorded from cell-attached and inside-out patches. Channels were initially recorded in the cell-attached configuration with both bath and pipette (6–20 MΩ) solutions containing (mM): NaCl, 144; KCl, 2.5; CaCl₂, 2.0; MgCl₂, 0.5; Hepes, 5; and glucose, 10; pH 7.4 with the addition of Tris base. Once in the cell-attached mode the superfusate was changed to one in which calcium had been omitted and magnesium increased to 5 mM. Patches were excised into a nominally Ca²⁺-free ‘internal’ solution (mM): KCl, 175; Hepes, 5; and BAPTA, 1; pH 7.2 with NaOH. To obtain a known free [Ca²⁺] (500 nM) to apply to the inside-out patches, the total amount of calcium that was added was predicted by the program REACT, version 2.01 (G. L. Smith, Department of Physiology, University of Glasgow, UK), taking account of the BAPTA concentration, pH, temperature and ionic strength. Channel activity was recorded using a patch-clamp amplifier (Axopatch 200A, Axon Instruments) and three criteria were applied to the channels to ensure M-channel-like activity: (i) a slope conductance of 12 or 7 pS, (ii) a low threshold for activation (near rest) and (iii) no obvious inactivation. A further test was to apply calcium to the inside-out patch (Selyanko & Brown, 1996a, b) using a motor-driven stepper device (solution exchange time, ∼10 ms). This same system was used to apply cADPR. Single channel data were acquired and analysed using pCLAMP software (version 6.0.3) (see Selyanko & Brown, 1996a, b, for details). Briefly, currents were recorded at room temperature using an Axopatch 200A patch-clamp amplifier, filtered at 1 kHz and digitized at 4 kHz. Currents were further filtered at 500 Hz for analysis.

Intracellular calcium measurements

Cells were incubated in fura-2 AM (5 μM) at 37°C in the dark for 10–45 min. Coverslips of cells loaded with fura-2 AM were placed on the stage of an inverted microscope (Nikon TMD) and superfused with the same solution used for the electrophysiology. Dual excitation (340 and 380 nm) of the cells was performed using a rota (Cairn) and the resultant emission (510 nm) was measured using a photomultiplier. Ratio signals were calculated on-line using PC-based software (Cairn). Ratio signals were not calibrated in terms of [Ca²⁺]_i; however, R_min was measured in a calcium-free solution (1 mM EGTA) and 100 μM BAPTA AM and R_max in 20 mM calcium, 50 μM ionomycin, which gave values of 0.21 and 4.70, respectively.

Drugs and chemicals

Cell culture media and supplements were obtained from Gibco unless otherwise stated. 8-Amino-cADPR was a gift of Professor A. Galione or supplied commercially. 8-Bromo-cADPR and fura-2 AM were from Molecular Probes. Acetylcholine chloride, bradykinin, Ruthenium Red, ATP, GTP and caffeine were obtained from Sigma. Ryanodine (high purity) was from Calbiochem. cADPR was from Amersham International plc. Buffer salts were from BDH. Drugs were applied to the superfusion system with the exception of 8-bromo-cADPR, 8-amino-cADPR and Ruthenium Red, which were applied via the recording pipette.

Data analysis

Suppression of I_K(M,ng) by acetylcholine was quantified by expressing the amplitude of the deactivation tail currents in the presence of acetylcholine as a percentage of that recorded just prior to its application. The means (±s.e.m.) of I_K(M,ng) inhibitions and amplitudes were compared using Student's t test (two tailed) for data in which only one comparison was made. However, if multiple comparisons were made with respect to a control value, then a one-way ANOVA was performed followed by the Dunnett multiple comparison test on data that gave P < 0.05 in the ANOVA. The outward current also elicited by acetylcholine was monitored; this occurred prior to the inward current (see Robbins et al. 1993) and an outward deflection greater than 100 pA from the holding current was used to indicate its presence. The data comparing the incidence of the outward current were analysed using a 4-fold contingency table. A less than 5 % level was considered significant. Intracellular concentrations of 8-amino-cADPR and 8-bromo-cADPR were calculated using the equation of Pusch & Neher (1988): τ = (r/r_o)³0.6R_aM^0.33, where τ is the time constant (in seconds) for cell dialysis, r is the radius of the NG108-15 cell (20 μm), r_o is the mean radius of chromaffin cells (7.68 μm), R_a is the access resistance (5 MΩ) and M is the molecular weight of the dialysing substance.

Channel open and closed states were identified on the basis of crossing the 50 % level of the channel current amplitude. Channel openings and closings shorter than 1 ms were ignored. Distributions of open and closed time were logarithmically binned and were fitted with exponential components by the method of maximum likelihood. Only events ≥ 1.5 ms were included in the fitting range. Open-point amplitude histograms were fitted with Gaussian curves.

Whole-cell current and single channel analysis were performed using commercially available software (Clampex, Clampfit, Fetchex, Fetchan and pStat, all supplied by Axon Instruments Inc).

RESULTS

Actions of cADPR antagonists

The presence of 100 μM 8-amino-cADPR in the patch pipette reduced the acetylcholine-induced inhibition of I_K(M,ng) in NG108-15 cells (Fig. 1A) compared with control cells. The time after establishing the whole-cell configuration at which the recordings of the inhibition were made was not significantly different between control (range, 12–24 min; mean ± s.e.m., 17.6 ± 1.4 min; n = 10) and 8-amino-cADPR-treated cells (range, 13–30 min; mean ±s.e.m., 18.8 ± 1.1 min; n = 16). The effect occurred at the three concentrations of acetylcholine tested (1, 10 and 100 μM). The reduction of inhibition occurred in a concentration-dependent manner at 10 and 100 μM 8-amino-cADPR (Fig. 1B). Although 8-amino-cADPR had a significant effect on the acetylcholine-evoked inhibition, it did not alter the initial amplitude of the I_K(M,ng) or the incidence of the outward current carried by Ca²⁺-activated K⁺ channels (I_K(Ca)) in response to phosphoinositide-mediated calcium release (Fig. 2).

Figure 1. 8-Amino-cADPR blocks acetylcholine-evoked inhibition of I_K(M,ng) in NG108-15 cells.

A, in the top example (i) inhibition is pronounced whereas in the condition where 8-amino-cADPR (100 μM) is present in the patch pipette (ii) the inhibition is reduced. I_K(M,ng) was recorded by holding the cells at a membrane potential of −20 mV and stepping to −50 mV for 1 s. B, pooled data showing concentration-inhibition curves for acetylcholine for control (^), intrapipette 8-amino-cADPR (10 μM; ▪) and intrapipette 8-amino-cADPR (100 μM; •). Means ± s.e.m. for 8–10 cells; statistical analysis gave * P < 0.05 and * * P < 0.01.

Figure 2. Lack of effect of 8-amino-cADPR on I_K(M,ng) amplitude and the incidence of acetylcholine-evoked outward current in NG108-15 cells.

A, mean and s.e.m.I_K(M,ng) amplitudes taken prior to acetylcholine application in the absence and presence of intrapipette 8-amino-cADPR. B, incidence of the acetylcholine-evoked outward current (I_IK(Ca)) measured as a percentage of total trials in the absence and presence of intrapipette 8-amino-cADPR. Number of cells indicated in parentheses.

In some experiments the development of the block of I_K(M,ng) inhibition with time was monitored (Fig. 3A). Acetylcholine was applied as quickly as feasibly possible after achieving whole-cell conditions (range, 1.5–4 min) and at a second time point 15–20 min later. In control cells the inhibition evoked by acetylcholine was not significantly different (70.1 ± 7.9 and 57.2 ± 9.3 %, respectively). However, the amount of inhibition at the second application of acetylcholine with either 10 or 100 μM 8-amino-cADPR in the pipette was reduced significantly (P < 0.01, t test) for both concentrations. There was the suggestion for the higher intracellular concentration that even after 3 min the response was already reduced (Fig. 3A).

Figure 3. Estimation of intracellular 8-amino-cADPR concentration using dialysis time.

A, mean and s.e.m. percentage I_K(M,ng) inhibition evoked by acetylcholine (100 μM) at two time points after achieving whole-cell status. Values given in control cells at 3 and 17 min. With 8-amino-cADPR in the pipette, time points were 3 and 19 min for 10 μM and 3 and 17 min for 100 μM. ** P < 0.01 when compared with the earlier time point for each concentration. Number of cells indicated in parentheses. B, using the dialysis time and pipette concentration, estimations were made of the intracellular concentrations of 8-amino-cADPR (see Methods) and plotted with respect to the relative inhibition. A logistic fit (Y = 100/(1 + (X/IC₅₀)^p)) is shown (continuous line): half-maximal concentration, 40 μM; slope (p), 0.74.

Intracellular dialysis of 8-amino-cADPR can be modelled by the equation derived by Pusch & Neher (1988; see Methods). With 10 μM 8-amino-cADPR in the pipette we calculated that at 3 and 19 min after breakthrough (the average time at which we tested the responses to acetylcholine), intracellular levels reached 3.3 and 9.3 μM, respectively. For the higher concentration (100 μM), 3 and 17 min after obtaining the whole-cell configuration, concentrations were 32 and 90 μM, respectively. Using these data we could therefore construct a concentration-inhibition curve (Fig. 3B) which when fitted to a logistic function (see Fig. 3 legend) gave a half-inhibition concentration of around 40 μM and a slope of 0.74.

As previously noted (Robbins et al. 1992, and authors' unpublished observations), the kinetics of the deactivation tails of M-like currents in NG108-15 cells are variable and frequently complex, often with biphasic or higher-order time constants. Since these might conceivably reflect current flow through different channels, we sought to assess whether 8-amino-cADPR had any selectivity with respect to inhibition of different components of the M-like current(s) by dividing the cells into two groups: those showing primarily fast deactivation kinetics (time constant of half-deactivation (τ_½) < 80 ms during 1 s steps), and those showing slower deactivation kinetics (τ_½ ≥ 80 ms). As shown in Fig. 4A, acetylcholine produced somewhat more inhibition in cells with time constants < 80 ms. Indeed, the inhibition for ‘fast’ currents was 80.3 ± 9.9 % (n = 6), which was statistically different (P < 0.05) from ‘slow’ currents, 43.5 ± 8.1 % (n = 6). 8-Amino-cADPR reduced the inhibition of fast-deactivating currents to a greater extent than it did the slow-deactivating currents, reaching statistical significance at 100 μM 8-amino-cADPR (Fig. 4B).

Figure 4. Acetylcholine and 8-amino-cADPR are more effective at modulating currents with fast deactivation kinetics than currents with slow deactivation kinetics.

A, in control cells I_K(M,ng) deactivation relaxation kinetics were measured (τ_½) and correlated to the amount of inhibition produced by 100 μM ACh. A regression line with a correlation coefficient, r, of −0.818 was fitted to the data (continuous line). B, I_K(M,ng) deactivation relaxations with a τ_½ of less than 80 ms were considered ‘fast’ and those equal to or greater than 80 ms were considered ‘slow’. The reduction in the inhibition mediated by 8-amino-cADPR was more pronounced in cells with fast deactivation kinetics than in those with slow deactivation kinetics. Number of cells given in parentheses; * P < 0.05.

Similar results were obtained with the less potent antagonist 8-bromo-cADPR, in that the inhibition of I_K(M,ng) by acetylcholine was significantly reduced at the higher concentration of 1 mM 8-bromo-cADPR without any significant change in I_K(M,ng) amplitude or incidence of the outward current (Fig. 5). Calculation of intracellular concentrations gave an estimated 40 % inhibition value of 900 μM (50 % reduction in the inhibition was not achieved) compared with an estimated 40 % value for 8-amino-cADPR of 20 μM (Fig. 3B).

Figure 5. Effect of 8-bromo-cADPR on I_K(M,ng) inhibition and amplitude, and the incidence of outward current.

Mean and s.e.m. inhibition (A), mean and s.e.m. amplitude (B) and incidence of acetylcholine-evoked I_IK(Ca) (C) for control cells and two concentrations of intrapipette 8-bromo-cADPR. Number of cells given in parentheses; *P < 0.05.

These results led us to conclude that a specific molecular target may mediate the action of cADPR with a specific binding site for cADPR. We have therefore further investigated the role of the established molecular target, the ryanodine receptor, in this response.

The role of ryanodine receptors

Application of low concentrations (≤ 1 μM) of ryanodine has been shown to stabilize the ryanodine receptor channel into a low conductance state leading to depletion of the calcium stores, whereas at higher concentrations (> 1 μM) the ryanodine receptor channel is blocked (see Sutko et al. 1997). In the presence of externally applied 1 or 10 μM ryanodine the acetylcholine-evoked inhibition of I_K(M,ng) was not significantly altered from controls. Similarly, neither the amplitude of the I_K(M,ng) nor the incidence of the outward current evoked by acetylcholine was affected (Fig. 6). We did, however, notice a small, slow increase in holding current (at −20 or −30 mV) after application of the lower concentration of ryanodine, which we attributed to slow release of calcium from the ryanodine receptor, which activated a proportion of the I_IK(Ca) present in these cells (Robbins, 1993). Further investigation of this event in a separate series of experiments, using fura-2 AM-loaded NG108-15 cells, showed that a small increase in intracellular calcium could be evoked by application of 1 μM ryanodine on some occasions (2 out of 7). However, the rise was less reliable and much smaller than that evoked by either 300 nM bradykinin (14 out of 17 cells tested) or 100 μM acetylcholine (18 out of 18 cells tested) (Fig. 7A). In comparison with the effect of low concentrations of ryanodine, a larger rise in intracellular calcium could be achieved with caffeine (10 mM) in 24 out of 51 cells, which was comparable in size to that seen with acetylcholine (Fig. 7B). Furthermore, the caffeine response was repeatable at least 4 times in the same cell. In 16 cells, which had previously responded to caffeine, preincubation with ryanodine (10 μM) blocked the caffeine-induced calcium rise in 14 and reduced it in two. On the other hand, preincubation with ryanodine did not inhibit the calcium rise mediated by bradykinin: all 15 cells tested responded under these conditions. These data confirmed that ryanodine was effective in our system and that there are indeed calcium stores that can be liberated by activation of ryanodine receptors.

Figure 6. Effect of ryanodine receptor modulators on I_K(M,ng) inhibition and amplitude, and the incidence of outward current.

s.e.m. ACh-evoked inhibition of I_K(M,ng) (A), mean and s.e.m. current amplitude (B) and incidence of ACh-evoked I_IK(Ca) (C). Data for control cells, two concentrations of bath-applied ryanodine (1 and 10 mM) as well as intrapipette Ruthenium Red (10 μM). Number of cells in parentheses. Although there was an increase in the incidence of outward current mediated by ACh in the presence of both ryanodine and Ruthenium Red, only the latter reached statisical significance (* P < 0.05).

Figure 7. Intracellular calcium concentration changes evoked by ryanodine, caffeine and acetylcholine in NG108-15 cells.

Fluorescence changes in a single fura-2 AM-loaded NG108-15 cell in response to ryanodine (1 μM) and acetylcholine (100 μM) (A) and changes in a separate cell in response to caffeine (10 mM) and acetylcholine (100 μM) (B).

Consistent with the above results, intracellular application of Ruthenium Red (10 μM), another ryanodine receptor antagonist, had little effect on the amplitude of I_K(M,ng) or the acetylcholine-evoked inhibition of I_K(M,ng) (Fig. 6). However, there was a significant increase in the proportion of cells demonstrating the outward current carried by I_K(Ca) (Fig. 6C). In other studies, intracellular dialysis of this concentration of Ruthenium Red completely suppressed caffeine-evoked calcium release in rat cerebellar Purkinje cells (Kano et al. 1995).

cADPR action on rat superior cervical ganglion cells

The action of cADPR on the M-current of rat superior cervical ganglion (SCG) cells was assessed in a manner similar to that for NG108-15 cells (Higashida et al. 1995). However, as has been demonstrated previously (Brown et al. 1995), under whole-cell recording conditions (needed to dialyse the cADPR) the amplitude of the M-current reduces rapidly (τ = 10 min). In our hands similar results were found, the time constant for ‘rundown’ in control cells of 11.4 ± 3.2 min (n = 6) being not significantly different from the rundown time constant with 5 μM cADPR in the pipette solution of 13.6 ± 4.0 min (n = 9). In comparison, the control rundown time constant in NG108-15 cells is of the order of 40 min (Higashida et al. 1995).

In view of the whole-cell results we tested the effect of cADPR applied directly to the cytoplasmic face of excised patches containing M-channels. Under these conditions SCG M-channel activity was stable for up to 30 min. Furthermore, M-channel activity recorded from membrane patches from rat SCG cells has advantages over patches from NG108-15 cells in that (a) the density of M-channels in NG108-15 cells is rather low for routine analysis and (b) the channels in NG108-15 cells are similar in terms of both their kinetics (at least in terms of the fast component) and their closure by bath application of muscarinic agonists in cell-attached mode (Selyanko et al. 1995).

At depolarized potentials (approximately 0 mV) and in the absence of Ca²⁺ in the bath (see Selyanko & Brown, 1996b), cell-attached patches of rat SCG showed a highly active potassium channel with little or no inactivation (Fig. 8A). On patch excision into the Ca²⁺-free solution (see Methods) the activity remained and application of 500 nM calcium inhibited the activity (Fig. 8B; and see Selyanko & Brown, 1996a). Application of 5 μM cADPR to the cytoplasmic face of the patch produced no noticeable change in the channel activity (Fig. 8C).

Figure 8. Effect of cADPR on M-channel activity in a membrane patch from a rat SCG neurone.

A, example of a cell-attached recording of single M-channel activity at 60 mV patch depolarization (membrane potential (V_m), ≈0 mV; Selyanko & Brown, 1996a, b). V_p, pipette potential. B and C, activity of the same channel recorded at 0 mV after excision of the patch into the Ca²⁺-free ‘internal’ solution (see Methods). The channel was inhibited by calcium (500 nM) applied for the duration of the horizontal line (B) but not by cADPR (5 μM) (C) applied to the cytoplasmic face in the absence of calcium. In C the activity obtained before (a) during (b) and after (c) application of cADPR is shown. All data were obtained from the same patch. For the effect of cADPR on the channel parameters see patch A in Table 1.

Similar results were found with a further two patches. Analysis of the channel current amplitude, open probability (P_o) and frequency of opening showed no consistent changes during the application of 5 μM cADPR for the three patches studied (Table 1). Two components in the open and two components in the shut time distributions (similar to those previously reported: Selyanko & Brown, 1996b) also remained unaltered.

Table 1.

Effect of cADPR on the parameters of M-channel activity recorded from inside-out membrane patches excised from rat SCG cells

	Control	cADPR (5 μs)	Wash
Patch A (1)
Amplitude (pA)	0.92	0.97	0.98
Po	0.89	0.86	0.89
Frequency (Hz)	42.7	33.5	49.4
Patch B (1)
Amplitude (pA)	0.86	0.82	0.86
Po	0.64	0.77	0.85
Frequency (Hz)	29.2	42.5	33.4
Patch C (2)
Mean current (pA)	1.10	1.24	1.60

Patches A (illustrated in Fig. 7) and B contained single M-channels allowing measurement of current amplitude, open probability (P_o) and frequency of opening. Patch C contained two channels, therefore the integral activity was expressed as the mean current through these channels measured over the period of 30 s of recordings under the different conditions. Number of channels given in parentheses.

DISCUSSION

cADPR antagonists

In investigating the action of antagonists of cADPR, we have further strengthened our case for the role of cADPR in I_K(M) inhibition. At 100 μM, 8-amino-cADPR can reduce acetylcholine-evoked I_K(M,ng) inhibition from over 50 % to around 10 % (Fig. 1). From estimations of achieved intracellular concentrations of 8-amino-cADPR the 50 % inhibition concentration is around 40 μM (Fig. 3). 8-Amino-cADPR was around 45 times more potent in blocking this I_K(M,ng) inhibition than 8-bromo-cADPR (Figs 3B and 5) based on comparisons of respective 40 % inhibition concentrations. This difference in potency is consistent with the potency difference between these two antagonists in blocking cADPR-evoked calcium release in sea urchin oocytes. In the oocyte system, 8-amino-cADPR is more potent than 8-bromo-cADPR by around 40 times (Lee, 1997). However, although the potency ratio is similar, the absolute values differ. For example, in the oocyte system 8-amino-cADPR and 8-bromo-cADPR inhibited cADPR-induced calcium release with IC₅₀ values of around 0.01 and 1.7 μM, respectively. The absolute potency values we have obtained are certainly open to question as we have not measured them directly, but the fact that they are 1000-fold higher than those needed to inhibit cADPR action on the ryanodine receptor may indicate that a different molecular target is involved in the modulation of I_K(M,ng). The action of these antagonists was selective in that the initial amplitude of I_K(M,ng) was not affected by the presence of these compounds, suggesting little or no basal inhibition of I_K(M,ng). Furthermore, the activation of the outward current generated by InsP₃-mediated calcium activation of I_K(Ca) channels in these cells (Robbins, 1993) was unaffected (Figs 2 and 5). These data lead us to suggest that m1 muscarinic receptor activation can in some way mobilize cADPR in NG108-15 cells. This is supported by the observation that m1 and m3 muscarinic receptor activation can increase ADP ribosyl cyclase activity in membrane preparations from NG108-15 cells (Higashida et al. 1997). However, at present little is known about the elements of the pathway that link muscarinic receptor activation with cADPR production. Previous work investigating the role of the nitric oxide-cyclic GMP (NO-cGMP) pathway in NG108-15 cells (Bowden & Robbins, 1997) has shown that the nitric oxide synthase (NOS) inhibitor (N^G-nitro-L-arginine, the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxaline-1-one, the nitric oxide donor sodium nitroprusside and the membrane-permeant cGMP analogue 8-bromo-cGMP have no effect on the M-current inhibition mediated by acetylcholine. This is in contrast to the situation in sea urchin eggs where the NO-cGMP pathway has been shown to mediate control of cADPR levels and ultimately calcium release (Galione et al. 1993).

Heterogeneity of I_K(M,ng)

In the light of the variation in deactivation kinetics and the differences both in the amount of inhibition evoked by acetylcholine and in the relative potency of 8-amino-cADPR on currents with fast and slow deactivation kinetics, more than one type of channel or subunit may be responsible for generating I_K(M,ng). Recent results have suggested that in order to achieve a current with kinetics and pharmacology similar to the M-current in SCG cells, two gene products need to be expressed, KCNQ2 and KCNQ3 (Wang et al. 1998). It would be interesting to test whether either of the currents generated by these subunits is inhibited by cADPR, and if so, whether there is a difference in their sensitivity.

Role of ryanodine receptors

The observation that I_K(M,ng) inhibition could not be perturbed by compounds that modulate or block ryanodine receptors suggests that, although ryanodine receptor-controlled calcium stores are present in NG108-15 cells (Fig. 7; and see Robbins et al. 1992) and cADPR can mobilize calcium from these stores in NG108-15 cells (Higashida et al. 1995), these receptors are not a prerequisite for cADPR action on the M-like current in these cells. Similar to what has been shown by others in NG108-15 cells (Empson & Galione, 1997), the calcium release evoked by cADPR can be modulated by ryanodine (H. Higashida, personal communication). We have shown that at low concentrations ryanodine can indeed activate ryanodine receptors in these cells and furthermore ryanodine at high concentrations can inhibit caffeine-evoked calcium release without affecting bradykinin-mediated responses. Intracellular dialysis of Ruthenium Red (10 μM) is very effective in blocking caffeine-induced responses in neurones of the same size order as the NG108-15 cells (Kano et al. 1995). The activation of calcium-dependent currents by acetylcholine was resistant to the cADPR antagonists ryanodine and Ruthenium Red, which is in agreement with the idea that the source of calcium for this event is mainly via an InsP₃ receptor-controlled calcium store (Brown & Higashida, 1988; Robbins, 1993). We did see a statistically significant increase in the incidence of outward current when Ruthenium Red was present in the pipette (Fig. 6C). Similarly, an increase (not reaching statistical significance) in the incidence of both the outward current evoked by acetylcholine and the calcium rise evoked by bradykinin was seen in the presence of ryanodine. Whether this reflects a kind of negative co-operativity between ryanodine and InsP₃ receptor-controlled calcium stores is not known.

It seems, then, that ryanodine receptor modulation is not a prerequisite for muscarinic receptor-evoked inhibition of I_K(M,ng), since acetylcholine-evoked receptor activation was equally effective in inhibiting the current in the presence of ryanodine receptor antagonists (Fig. 6). This is not to say that calcium is not involved in the inhibition of receptor-mediated inhibition of I_K(M,ng), as recent studies have demonstrated the M-channel to be sensitive to calcium in SCG cells (Selyanko & Brown, 1996a). However, the fact that a rise in calcium is not flurometrically detectable in SCG cells after muscarinic receptor stimulation and that activation of I_K(Ca) and inhibition of I_K(M,ng) in NG108-15 cells are separable (Robbins et al. 1993) does leave the role of calcium still open to question. However, other membrane currents modulated by cADPR have also been shown to be insensitive to ryanodine receptor antagonists (Currie et al. 1992).

cADPR and M-channel activity

We have shown that application of cADPR directly to the cytoplasmic face of SCG cells did not reduce or alter M-channel activity (Fig. 8), whereas whole-cell dialysis in NG108-15 cells can reduce the whole-cell current (Higashida et al. 1995). This apparent contradiction can be explained by a number of possibilities. Firstly, that the channels that carry the M-current in these two cell types are different; however, as far as has been studied the channels show more similarities than differences (Selyanko et al. 1995). Secondly, that an intervening element(s) is required for cADPR activity, which is lost on patch excision. Thirdly, that more than one diffusable messenger is required, acting in concert to inhibit the channel activity.