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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 μm) evoked a muscarinic cationic current (mICat) as small as ∼10% of mICat in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein–cationic channel coupling. The M3-KO and WT mICat each showed a U-shaped current–voltage (I–V) relationship, whereas the M2-KO mICat displayed a linear I–V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times Oτ= 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (Oτ= 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (Oτ= 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I–V curve similar to the WT mICat. The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mICat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.

In visceral smooth muscles including those of the gastrointestinal tract, M2 and M3 muscarinic receptors are coexpressed with a preponderance of the former subtype (M2: M3= 3–5: 1; Caulfield, 1993; Eglen et al. 1996). These receptors mediate the actions of the parasympathetic neurotransmitter acetylcholine or muscarinic agonists to produce excitation and contraction (Bolton, 1979). The M2 receptor preferentially couples to Gi/o-type G-proteins which mediate the inhibition of adenylyl cyclase. On the other hand, the M3 receptor interacts with Gq/11 type G-proteins, stimulating phospholipase Cβ (PLCβ) and hydrolysis of phosphatidylinositol 4,5-bisphospate (PIP2), which results in production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (see Ehlert et al. 1997, 1999). Besides these biochemical effects, muscarinic receptor stimulation also causes the opening of cationic channels with less selectivity for various monovalent cations (Kuriyama et al. 1998; Bolton et al. 1999; Sanders, 2001; Unno et al. 2003). Under physiological ionic conditions, the muscarinic receptor-mediated cationic current (mICat) is carried mainly by Na+ with a reversal potential between −10 and 0 mV, whereby depolarization is produced and so the discharge of action potentials and slow-wave potentials initiated or accelerated unless the depolarization is too strong (Bolton, 1972; So & Kim, 2003; Unno et al. 2003). These electrical events result in increased Ca2+ influx via voltage-dependent Ca2+ channels, providing Ca2+ for contraction. Thus, the generation of mICat is a most important mechanism underlying the contractile response.

Therefore, the signal transduction and channel gating mechanisms involved in mICat generation have been studied in various visceral smooth muscles (see Unno et al. 2006b). Studies with guinea-pig ileum using classical pharmacological tools have led to the hypothesis that a unique signal transduction mechanism involving both M2 and M3 receptors is largely responsible for generation of mICat (Zholos & Bolton, 1997; Okamoto et al. 2002). In this pathway, the M2 and M3 receptors provide concurrent, but different, signals for cationic channel opening via coupling to Go protein and a PLC isozyme, respectively (Komori et al. 1992, 1998; Zholos & Bolton, 1997; Yan et al. 2003), and PLC activity seems to be independent of Gq/11 regulation, and DAG or Ca2+-store depletion does not significantly participate in channel opening (Okamoto et al. 2004; Zholos et al. 2004a), although IP3 is suggested to have some potentiating roles in channel opening (Gordienko & Zholos, 2004). Meanwhile, single channel recordings identified three types of muscarinic cationic channel with unitary conductances of 10, 60 and 130 pS and showed that the medium conductance channel (60 pS) mediates the major part of whole-cell mICat (Zholos et al. 2004b). The precise roles of M2 and M3 receptors in activation of these cationic channels remain to be elucidated. Moreover, the possible synergism between M2 and M3 receptors needs to be tested more rigorously.

A promising approach to address these issues is the use of mutant mice deficient in certain subtypes of muscarinic receptor (Unno et al. 2005, 2006a; and also see Wess, 2004). Nonetheless, relatively little is known about mICat in murine intestinal myocytes. We recently studied carbachol-induced mICat responses and found that murine mICat shares similar signal transduction mechanisms with guinea-pig mICat (Sakamoto et al. 2006). Non-stationary noise analysis of whole-cell mICat also suggested that the major channels mediating mICat have almost the same unitary conductance in the two species (Sakamoto et al. 2006). Dresviannikov et al. (2006), using single channel recording techniques, revealed muscarinic 17-, 70- and 140-pS cationic channels with the 70-pS channel having the major role in mediating whole-cell mICat. There was again a substantial similarity between mouse and guinea-pig channel properties (see above).

In the present study, we have used M2 or M3 single knockout (KO) and M2/M3 double-KO mice as experimental tools, aiming to identify muscarinic signalling pathways leading to mICat generation and target channels for the different pathways and to elucidate the role of M2 and M3 receptors in these processes. Our data from whole-cell and outside-out patch experiments strongly support the idea that intact gut myocytes are endowed with three distinct muscarinic pathways linked to cationic channel activation; two of these involve either M2 or M3 receptors and the third one requires the presence of both receptor subtypes. The M2- and M3- pathways target the 70-pS channel and both 70 and 120-pS channels, respectively. The M2/M3 pathway targets the 70-pS channel and causes much longer channel openings than the other two pathways, playing a major role in inducing whole-cell mICat.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

All procedures described below were performed according to the guidelines approved by the local animal ethics committee of the Faculty of Applied Biological Sciences, Gifu University.

Animals and cell preparation

The generation of the M2 and M3 muscarinic receptor single KO and M2/M3 double-KO mice has been previously described (Gomeza et al. 1999; Yamada et al. 2001; Struckmann et al. 2003). The genomic background of the mice used in the present study was 129J1 (50%) × CF1 (50%) for the M2-KO and their corresponding wild-type (WT) mice, 129SvEv (50%) × CF1 (50%) for the M3-KO and their corresponding WT mice, and 129J1 (25%) × 129SvEv (25%) × CF1 (50%) for the M2/M3 double-KO mice. Animals were housed in polycarbonate ventilated cages. The animal room was maintained at 22–25°C with a relative humidity of 40–60% and a daily light–dark cycle (07.00–19.00 h light). Food (MF or CMF; Oriental Yeast Co., Japan) and water were supplied ad libitum. The two WT strains of mouse were used without distinction for experiments (Unno et al. 2006a).

Mice of either sex weighing 30–40 g were killed by cervical dislocation. A gut segment of 15 cm in length was removed from a region over the jejunum and ileum, and cut into 1.5–2.0 cm pieces, from each of which the longitudinal smooth muscle layer was carefully peeled off. Single smooth muscle cells were isolated from the muscle layers enzymatically, as described elsewhere (Sakamoto et al. 2006). The cells were suspended in physiological salt solution (PSS; for composition, see below) containing 0.5 mm CaCl2, placed on coverslips in small aliquots and stored at 4°C until use on the same day.

Electrophysiological recordings

Membrane current recordings were made at room temperature (22–26°C) using conventional whole-cell or outside-out patch clamp techniques with glass patch pipettes of 3–7 MΩ. The tip of the pipettes used for outside-out patch recordings was coated with Silpot 184W/C elastomer (Dow Corning, Asia, Ltd, Kanagawa, Japan). A CEZ-2300 voltage-clamp amplifier (Nihon Kohden, Tokyo, Japan) was used for whole-cell recording, and current signals were filtered at 1 kHz and captured at a sampling rate of 4 kHz using an analog–digital converter (DIGIDATA 1322A; Axon Instruments, Inc., Union City, CA, USA) interfaced to a computer (IMC-P642400, Inter Medical Co, Nagoya, Japan) running the pCLAMP program (version 9, Axon Instruments). When current–voltage (I–V) relationship for mICat was investigated, current signals were also stored on a digital tape (DM120D; Hitachi Maxell, Tokyo, Japan) with a digital tape recorder (RO-101; TEAC, Tokyo, Japan) for later analysis and illustration. An Axopatch 200B voltage-clamp amplifier (Axon Instruments) was used for the recording of single channel currents in outside-out patches, and current signals were filtered at 2 kHz and captured at a sampling rate of 50 kHz. For illustration, the current signals were filtered with a 0.5 or 1 kHz lowpass Gaussian filter. An outside-out patch was excised from the cell after whole-cell mICat had fully developed in response to carbachol, as described by Zholos et al. (2004b). In some cases, outside out patches were formed before carbachol application. Every patch tested was excised from a different cell.

When changes in membrane potential were measured, single cells were held under current clamp mode with nystatin-perforated patch clamp techniques, as previously described (Kohda et al. 1997). Signals were stored in the same way as described for whole-cell current recordings.

Solutions

The external solution in which whole-cell and outside-out patch cationic currents were recorded had the following composition (mm): CsCl 120, glucose 12 and Hepes 10, with pH adjusted to 7.4 with CsOH (Cs+= 134 mm in total). Patch pipettes were filled with solution of the following composition (mm): CsCl 80, MgATP 1.0, Na2GTP 1.0, creatine 5, glucose 20, Hepes 10, BAPTA 10 and CaCl2 4.6 (calculated free calcium ≈ 100 nm), with pH adjusted to 7.4 with CsOH (Cs+= 142 mm in total, the calculated equilibrium potential for Cs+ in the external and internal solutions was ∼1.5 mV). GTP was omitted when GTPγS was applied intracellularly via patch pipettes. The Cs+-rich solutions served to block K+ currents and prevent cationic currents from being modulated by changes in cytosolic Ca2+ concentration ([Ca2+]i) (Sakamoto et al. 2006).

For whole-cell recording of Ca2+-activated K+ current (IK-Ca), cells were bathed in PSS consisting of (mm): NaCl 126, KCl 6, CaCl2 2, MgCl2 1.2, glucose 14 and Hepes 10.5, with pH adjusted to 7.2 with NaOH; they were dialysed intracellularly with a pipette solution consisting of (mm): KCl 134, MgCl2 1.2, MgATP 1.0, Na2GTP 0.1, EGTA 0.05, glucose 14 and Hepes 10.5, with pH adjusted to 7.2 with KOH (Komori et al. 1992).

In measurement of membrane potential changes, normal PSS was used as the bath solution and a K+-rich solution used as pipette solution that consisted of (mm): KCl 134 and Hepes 10.5 (adjusted to pH 7.4 with KOH), containing nystatin at 0.2 mg ml−1 (Kohda et al. 1998).

Analysis of current signals

pCLAMP 9 (Axon Instruments) and Origin 7.0 software (OriginLab Corp., Northampton, MA, USA) were used for analysis and plotting of data. Relationships of mICat to membrane potential were obtained by applying a 5 s ramp pulse from −120 to 40 mV before and after carbachol application, and I–V curves were constructed after leakage subtraction. Whole-cell membrane capacitances were estimated from capacitative currents evoked upon a hyperpolarizing 10 mV step. Single-channel events in outside-out patches were detected on the basis of the half-amplitude threshold-crossing criterion, in which events < 0.3 ms (e.g. about twice the rise time for 2 kHz filter) were ignored (Zholos et al. 2004b). For constructing amplitude histograms, fitted-levels amplitudes, instead of all-point amplitudes, were plotted with 0.1 pA bins, because some drifts in the baseline occurred in long time recordings and there were many openings that were too short to reach full amplitude in some patches, so that the resultant amplitude distributions were smeared (Colquhoun, 1994). Amplitude histograms of channel events from a 10–115 s recording were subjected to Gaussian fitting in order for single channel current amplitudes to be determined. Histograms of channel open-times were constructed with 0.1 ms bins and fitted conventionally with one or two appropriate exponential functions by the method of maximum likelihood. When three or more exponential components were needed to fit, the open-time histograms were constructed as a distribution of the logarithm of the open-time (20 bins per decade) using the exponential log probability function in pCLAMP software (Colquhoun, 1994). The Y-axis was represented by the frequency density, which is the square root of the number of events. If the activity of different single channels in unitary current size occurred in the same membrane patch (Figs 6B and 9C), channel events between ± 1.5 s.d. of the mean in their respective amplitude histograms were used to determine ‘apparent mean open-times (Oτ)' and calculate the open probability (Po) for the different single channels. Po was calculated using the following equation

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Figure 6. Carbachol activates 70- and 120-pS channels in bursting and brief opening mode in the outside-out patches excised from M2-KO cells After establishment of whole-cell clamp mode at −40 mV, mICat was activated by carbachol (100 μm), and then the membrane patch was excised to form outside-out configuration in the presence of the agonist. A, typical example of single channel current evoked by carbachol in the absence and presence of atropine (3 μm). Time-expanded, consecutive traces corresponding to a and b are shown in the lower panels. The asterisks indicate opening of the 20-pS channel. B, fitted levels amplitude histogram counted per 0.1 pA bins for the channel currents. The distribution was fitted by the sum of three Gaussian functions, where peak amplitudes of 2.7 and 4.3 pA were detected. C, open time histograms for the 70-pS (left hand panel) and 120-pS (right hand panel) channels. The open time distributions per 0.1 ms bins for both channels were fitted by the one exponential component with the time constants of 0.4 ms (for 70-pS channel) and 0.3 ms (for 120-pS channel), respectively.

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Figure 9. Amplitude and open time analysis for the 70-pS and 120-pS channels detected in WT patches A and B, fitted levels amplitude (A) and open time (B) histograms for the channel current shown in Fig. 8A. Gaussian function fitting to the amplitude histogram gave a peak amplitude of 3.1 pA as a major component. The open time distributions were plotted as log open time (20 bins per decade scale) versus frequency (square root scale) and fitted by the sum of four exponential components with the time constants of 0.3 (Oτ1), 1.8 (Oτ2), 10.0 (Oτ3) and 97.6 ms (Oτ4). C and D, fitted levels amplitude (C) and open time (D) histograms for the channel current shown in Fig. 8B. In C, Gaussian function fitting to the amplitude histogram gave peak amplitudes of 2.8 and 5.0 pA as major components. In D, the open time distributions for 70-pS (left hand panel) and 120-pS (right hand panel) channels were fitted by the one exponential component with the time constants of 0.3 ms and 0.2 ms, respectively.

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cAMP measurements

Measurements of cAMP were made basically according to the method of Kitazawa et al. (2000). In brief, isolated mouse ileal longitudinal muscle layers weighing approximately 5–10 mg were prepared and suspended in warmed (37°C) and gassed (95% O2–5% CO2) Krebs solution (mm: NaCl 118.4, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and glucose 11.5) containing the phosphodiesterase inhibitor IBMX (1 mm). To investigate the effect of carbachol on isoprenaline-induced increases in cyclic AMP levels, the muscle strips were incubated for 10 min in the presence or absence of carbachol (1 μm). After this incubation, isoprenaline (1 μm) was added and allowed to act for 5 min, and then the muscle strips were quickly frozen in liquid nitrogen and homogenized in 6% trichloroacetic acid solution with a Polytron. The homogenate was centrifuged at 2000 g for 20 min (twice) and the resulting supernatant was collected. The pellet was dissolved in 0.1 n NaOH for protein determination by the BCA protein assay kit (Pierce, Rockford, IL, USA). After removing trichloroacetic acid in the supernatant by washing three times with water-saturated ether, cAMP in the extract was assayed using an enzyme immunoassay kit (Amersham International Ltd, Piscataway, NJ, USA). Tissue cyclic AMP levels were expressed as picomoles per milligram protein.

Chemicals

Carbamylcholine chloride (carbachol), 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), guanosine 5′-o-(3-thiotriphosphate) tetralithium salt (GTPγS), 3-isobutyl-1-methylxanthine (IBMX), prostaglandin F2α (PGF2α) and isoprenaline were purchased from Sigma (St Louis, MO, USA). Caffeine, atropine sulphate, nystatin, quinidine sulphate and 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17yl]amino]-1H-pyrrole-2,5-dione (U73122) were purchased from WAKO (Tokyo, Japan).

Statistics

Values in the text are given as means ±s.e.m. with the number of measurements. Statistical significance between two groups was assessed using Student's unpaired t test. When more than three groups were compared, one-way analysis of variance (ANOVA) followed by a post hoc Bonferroni-type multiple comparison test was used. Differences were considered significant when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Muscarinic pathways mediating cationic currents

Muscarinic cationic currents (mICat) induced by carbachol were measured in single intestinal smooth muscle cells from WT and muscarinic receptor mutant mice. Experiments were performed under conditions optimal for activation and isolation of mICat (nearly symmetrical Cs+ solutions and [Ca2+]i buffered to ∼100 nm; Sakamoto et al. 2006). Application of a maximally effective carbachol concentration (100 μm) at a holding potential of −50 mV produced mICat of 209.4 ± 22.2 pA (n= 17) in cells from the WT strain (Fig. 1A). The current amplitude resembled that obtained previously in the same type of cells from a conventional strain of mouse (233 pA; Sakamoto et al. 2006). Carbachol (100 μm) also evoked mICat in M2-KO or M3-KO cells but current amplitudes were as small as 24.0 ± 3.3 pA (n= 15) or 12.9 ± 1.4 pA (n= 21), respectively (Fig. 1A and C). The differences in current size did not involve cell size, since the whole-cell membrane capacitances of M2-KO cells (37.4 ± 3.2 pF, n= 15) and M3-KO cells (39.0 ± 2.2 pF, n= 15) resembled the corresponding WT value (40.5 ± 2.6 pF, n= 15). The current response of M2-KO or M3-KO cells was blocked by atropine (1 μm), indicating a muscarinic receptor-mediated response. Strikingly, carbachol was unable to induce an appreciable current in cells from the M2/M3 double-KO strain (Fig. 1A and C), indicating that no other muscarinic receptor subtypes, besides M2 and M3, participate in generation of mICat. Hence, the mICat responses in the M2-KO and M3-KO cells are mediated by M3 and M2 receptors, respectively; however, their respective amplitudes were 11 and 6% of the WT value (Fig. 1C), indicating that the WT mICat is not a simple mixture of M2 and M3 receptor responses.

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Figure 1. The simultaneous presence of both M2 and M3 receptors is required for full activation of mICat in longitudinal smooth muscle cells of mouse small intestine Membrane current responses were recorded using conventional whole-cell voltage-clamp techniques under conditions where Cs+ was used as the major cation outside and inside of the cell (∼140 mm). The intracellular Ca2+ concentration ([Ca2+]i) was clamped with a BAPTA/Ca2+ buffer ([Ca2+]I≈ 100 nm). A, mICat responses to carbachol at the holding potential of –50 mV in WT, M2-, M3- or M2/M3 double-KO cells. Dashed lines indicate 0 current levels. B, the direct G protein activator GTPγS, which was applied via the patch pipette (▴), evoked an mICat-like current irrespective of mouse genotype. C, summary of carbachol- or GTPγS-induced mICat amplitudes. *Significantly different (P < 0.001) from the corresponding WT value.

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The direct G protein activator GTPγS has been shown to induce cationic currents in guinea-pig ileal and gastric myocytes which are very similar to mICat in essential pharmacological and biophysical properties (Chen et al. 1993; So & Kim, 2003; Zholos et al. 2004b). As shown in Fig. 1B, GTPγS (200 μm) injected into cells via a patch pipette induced an inward current in cells from WT and all three mutant strains. The inward current developed gradually to reach a steady level within 3–10 min, and quickly disappeared when quinidine (a cationic channel blocker; Kim et al. 1995) or U73122 (a PLC inhibitor; Zholos et al. 2004a; Sakamoto et al. 2006) was added (Fig. 1B). The amplitudes of GTPγS-induced currents that had reached a steady level did not significantly differ between the four mouse strains or any pair of them (Fig. 1C). This observation suggests that the lack of M2 and/or M3 receptors does not affect G protein–cationic channel links or cationic channel expression.

To examine the efficiency of muscarinic receptor/G protein coupling in the cells from the different mutant strains, we carried out a series of additional experiments. It is well documented that M3 receptor activation links via the Gq/11-PLCβ system to intracellular Ca2+ release (Eglen et al. 1993; Ehlert et al. 1997). Therefore, under appropriate recording conditions (see Methods), the M3-mediated Ca2+ release events were monitored as Ca2+-activated K+ current (IK-Ca). At a holding potential of 0 mV, spontaneous transient outward currents (STOCs) of IK-Ca (Benham & Bolton, 1986) due to integral Ca2+ sparks (Bolton & Gordienko, 1998) occurred with various frequencies and amplitudes. The discharge activity of STOCs seemed comparable between WT and all three mutant strains. In M2-KO cells, carbachol (100 μm) evoked a brief IK-Ca (1.2 ± 0.1 nA, n= 5) that was similar in size to that observed in WT cells (1.6 ± 0.3 nA, n= 8) (Fig. 2A and B). Following the IK-Ca response, STOC discharge ceased, and subsequent application of the potent Ca2+ releaser caffeine (10 mm) was without effect, indicative of carbachol-mediated Ca2+ store depletion. As expected, carbachol had no effect in M3-KO or M2/M3 double-KO cells, as the STOC discharge remained almost unchanged, but subsequent caffeine application evoked a prominent IK-Ca in both M3-KO and M2/M3 double-KO cells (1.7 ± 0.6 nA and 1.4 ± 0.3 nA, respectively; n= 7) (Fig. 2A and B).

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Figure 2. M3 receptor-mediated intracellular Ca2+ release and M2 receptor-mediated inhibition of adenylyl cyclase are fully operative in M2-KO and M3-KO smooth muscle cells, respectively A, carbachol- or caffeine-induced Ca2+ release events detected as IK-Ca at the holding potential of 0 mV in WT, M2-, M3- or M2/M3 double-KO cells. B, summary of carbachol- or caffeine-induced IK-Ca amplitudes. C, basal and isoprenaline (1 μm)-stimulated cAMP levels in ileal longitudinal muscle tissues from WT, M2-KO, M3-KO and M2/M3 double-KO mice. D, carbachol (1 μm)-mediated changes in cAMP levels in WT, M2-KO, M3-KO and M2/M3 double-KO preparations. Measurements were carried out in the presence of 1 μm isoprenaline. *Significantly different (in B: P < 0.01, in D: P < 0.05) from the corresponding WT value.

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We also examined effect of carbachol on intracellular cAMP levels, since the existence of the M2 receptor–Gi/o–adenylate cyclase system is well-documented (Candel et al. 1990; Griffin & Ehlert, 1992). To be able to detect M2 receptor-mediated decreases in cAMP levels, assays were carried out in the presence of a stimulatory concentration of isoprenaline (1 μm), a β-adrenergic receptor agonist. There were no significant differences in basal or stimulated cAMP levels among tissue preparations from the mutant and WT strains (Fig. 2C). Carbachol (1 μm) inhibited the isoprenaline-induced increase in cAMP in the M3-KO but not in the M2-KO or M2/M3-KO tissues (Fig. 2D). The extent of inhibition observed with the M3-KO tissues (49.7 ± 14.0%, n= 5) was greater than that found with the WT tissues (26.1 ± 3.9%, n= 8).

Taken together, these data clearly indicate that the M2 receptors expressed in the M3-KO cells as well as the M3 receptors expressed in the M2-KO cells are efficiently coupled to their respective G protein/effector systems.

The results summarized in Figs 1 and 2 clearly demonstrate the existence of three distinct pathways leading to mICat generation, two of which involve either M2 and M3 receptors and the third requires the presence of both receptor subtypes. The latter pathway (M2/M3 pathway) may mediate the major part of mICat in WT gut myocytes, at least in symmetrical Cs+ solutions with [Ca2+]i buffered to ∼100 nm.

I–V relationship and Ca2+ sensitivity of mICat responses

Voltage dependency and Ca2+ sensitivity of mICat were investigated in M2-KO, M3-KO and WT cells. A negative-going voltage ramp pulse from +40 to –120 mV (over 5 s) was applied to cells stimulated with 100 μm carbachol, and I–V relationships for mICat were obtained after leakage subtraction. As shown in Fig. 3, when net mICat was plotted against potential, the resulting curves were U-shaped in the WT cells due to deviation from an ohmic conductance at negative potentials, as previously observed in ileal cells from the conventional strain of mouse (Sakamoto et al. 2006). I–V curves were similarly U-shaped in the M3-KO cells, but roughly linear in the M2-KO cells. Independent of mouse genotype, the reversal potential (Erev) for mICat was close to 0 mV. These observations suggest that cationic channels activated via the M2- and the M2/M3 pathways are similar in their intrinsic voltage dependency.

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Figure 3. The current–voltage (I–V) relationships of mICat in WT (A), M2-KO (B) and M3-KO (C) cells The upper panels show typical examples of carbachol (100 μm)-evoked mICat, where a negative going ramp pulse from +40 mV to −120 mV over 5 s was applied before and after application of carbachol. The lower panels show the corresponding I–V curves constructed by leakage subtraction. Dashed lines in the upper panels indicate 0 current levels.

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We also tested the Ca2+ sensitivity of mICat under conditions where cells were bathed in PSS and filled with a Cs+-rich pipette solution not containing the BAPTA/Ca2+ mixture but with most chloride replaced with glutamate in order to prevent possible generation of Ca2+-activated chloride currents (Sakamoto et al. 2006). Under these conditions, voltage-gated Ca2+ influxes were evoked by a 0.2 s depolarizing pulse in the presence of 100 μm carbachol. As shown in Fig. 4A, in WT cells, a large inward current of 200–500 pA appeared upon repolarization following the depolarizing pulse, and over 3–10 s later the tail current declined to a current level during the agonist application. After application of 1 μm carbachol which evoked a very weak mICat, a tail current was clearly observed (data not shown). In contrast, M2-KO or M3-KO cells exhibited no appreciable tail current upon repolarization following the depolarizing pulse in the presence of carbachol (100 μm) (Fig. 4B and C). These observations suggest that Ca2+ has no potentiating role in channel activation in the M2 and M3 pathways.

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Figure 4. Inward tail current responses evoked by Ca2+ entry through voltage-gated Ca2+ channels in WT (A), M2-KO (B) and M3-KO (C) cells Voltage-gated Ca2+ currents were evoked by voltage steps to 0 mV for 0.2 s from the holding potential of −50 mV in the absence (a) and presence (b) of carbachol (100 μm). The time-expanded traces of Ca2+ currents a and b are shown in the right hand panel. Dashed lines indicate 0 current levels. An inward tail current appeared only in WT cells.

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Membrane potential responses

To assess the physiological relevance of the three mICat-inducing pathways, we observed changes in membrane potential produced by carbachol in cells from the four different mouse strains, using nystatin-perforated patch-clamp techniques (Kohda et al. 1997). The cells were bathed in PSS, dialysed with a K+-rich pipette solution, and held under current-clamp mode. Under such equi-physiological conditions, the cells had a resting potential between −40 and −60 mV, and there was no significant difference in the averaged resting potential among those mouse strains (WT: –46.3 ± 1.3 mV, n= 14; M2-KO: −50.7 ± 1.5 mV, n= 16; M3-KO: −47.7 ± 1.9 mV, n= 16; M2/M3 double-KO: –44.6 ± 2.5 mV, n= 5). In some cells (about 20% in each strain), spontaneous discharges of the action potential were seen. In WT cells, carbachol at 0.1–0.3 μm produced a moderate depolarization of 10–20 mV and generation or acceleration of spike activity (left panel in Fig. 5A). Higher concentrations of carbachol (> 1 μm) evoked a full depolarization (41.8 ± 1.5 mV in amplitude, n= 4) such that the membrane potential reached near-equilibrium potential (−10 mV) reported for the muscarinic depolarization in physiological ionic gradients (Bolton, 1972; Benham et al. 1985; Unno et al. 2000) (Fig. 5A and E). In M2-KO cells, 0.1 μm carbachol was almost without effect, but 1 μm induced excitatory events as seen in WT cells stimulated with 0.1 μm carbachol. At higher carbachol concentrations of 30 or 100 μm, a full depolarization was evoked (Fig. 5B). In M3-KO cells, carbachol was usually ineffective at 1–3 μm, but sometimes fluctuations in resting potential occurred with a small sustained depolarization. At 30 or 100 μm, a significant depolarization of about 10 mV was evoked, on which increased spike activity was superimposed (Fig. 5C and E). M2/M3 double-KO cells were insensitive to carbachol (up to 100 μm), but did respond to prostaglandin F (PGF) by a depolarization of 20–30 mV (Fig. 5D). These observations suggest that the three muscarinic pathways are relevant in depolarizing the membrane with a rank order of M2/M3 > M3 > M2, similar to that for mICat generation.

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Figure 5. The M2/M3-operated pathway is more efficient in producing membrane depolarization and increased spike activity than the M2- or M3-operated pathways A–D, membrane potential responses to carbachol (0.1–100 μm) recorded using nystatin-perforated patch-clamp techniques in WT (A), M2-KO (B), M3-KO (C) or M2/M3 double-KO (D) cells. Dashed lines indicate 0 mV level. In D, PGF was applied in the presence of carbachol. E, summary of carbachol-induced depolarization responses. *Significantly different (P < 0.001) from the corresponding WT value.

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Identification of target channels for the muscarinic pathways

Channel activity was observed in outside-out patches that were excised from M2-KO, M3-KO or WT cells held at –40 mV and exposed to 100 μm carbachol, as described in guinea-pig ileal myocytes (Zholos et al. 2004b). Channel current recordings were made in nearly symmetrical Cs+ solutions and [Ca2+]i buffered to ∼100 nm. In this series of studies, we found three types of muscarinic cationic channels with different unitary conductances of ∼20, ∼70 and ∼120 pS. However, the 20-pS channel activity was small in unitary current (∼1 pA) and very low in opening frequency (e.g. see asterisks in Figs 6–8); it therefore seemed to contribute little to whole-cell mICat (Zholos et al. 2004b; Dresviannikov et al. 2006). Therefore, we did not study the 20-pS channel activity in any further detail in the present study.

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Figure 7. Carbachol activates only the 70-pS channel with a longer open state in the outside-out patches from M3-KO cells A, typical example of single channel current evoked by carbachol (100 μm) in the absence and presence of atropine (3 μm). Time-expanded, consecutive traces corresponding to a and b are shown in the lower panels. The asterisks indicate opening of the 20-pS channel. B, fitted levels amplitude histogram for the channel current fitted by Gaussian functions where peak amplitude of 2.9 pA was detected as a major component. C, open time histogram for the 70-pS channel. The open time distributions were plotted as log open time (20 bins per decade scale) versus frequency (square root scale) and fitted by the sum of three exponential components with the time constants of 0.2 (Oτ1), 1.6 (Oτ2) and 13.7 ms (Oτ3).

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Figure 8. Single channel activity evoked by carbachol in the outside-out patches from WT cells Three typical examples of carbachol (100 μm)-activated single channel currents. A, 70-pS channel activity having a very long-open state. B, 70-pS and 120-pS channel activities with a short-open state resembling those seen in M2-KO patches. C, 70-pS and 120-pS channel activities consisting of mixed pattern of M2-KO and M3-KO patches. In A and B, current traces after the application of quinidine (10 μm, A) or atropine (3 μm, B) are also shown in the lower panel. In C, time-expanded traces corresponding to a and b are shown in the lower panels. * and • indicate the opening of the 20-pS and 120-pS channels, respectively.

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Of 34 outside-out patches excised from M2-KO cells, 29 (85%) had no significant channel activity beyond the basal noise level, but the remaining 5 (15%) were clearly active, as exemplified in Fig. 6A. The channel activity consisted of bursts of brief openings (Fig. 6Aa), and the ongoing channel activity was terminated upon addition of atropine (3 μm) in the bath solution containing carbachol (Fig. 6Ab, n= 3). Figure 6B shows current amplitude histograms for the channel activity, which revealed two major peaks at 2.7 and 4.3 pA. The amplitudes of unitary current represented by these major peaks were 2.7 ± 0.1 and 4.7 ± 0.1 pA (n= 5) and single channel conductances estimated from the unitary current amplitudes at Erev= 0 mV were 68.6 ± 1.2 pS and 117.1 ± 3.2 pS (n= 5), respectively. The probabilities of channel opening (Po: see Methods) varied from 0.01 to 0.32 (0.08 ± 0.06 on average, n= 5) for the 70-pS and from 0.002 to 0.09 (0.02 ± 0.02 on average, n= 5) for the 120-pS channel. Although the Po for the 70-pS channel was relatively great, this value might have been overestimated due to limitations of the recording system, in which some brief, abortive 120-pS channel currents might be counted as open events of the 70-pS channels. Figure 6C shows histograms of distribution of apparent open-times for the 70-pS and the 120-pS channel detected by analysis in Fig. 6B. The individual histograms followed a single exponential function with a time constant (i.e. mean apparent open time; Oτ) of 0.42 ms for the 70-pS channel and 0.27 ms for the 120-pS channel. Averaged Oτ values for the respective channels were 0.55 ± 0.12 ms (n= 5) and 0.23 ± 0.02 ms (n= 3). In the two patches, the number of acquired events for the 120-pS channel was not enough for the fitting process. The results indicate that the M3 pathway targets both 70-pS and 120-pS channels.

Out of 25 patches from M3-KO cells, 22 (88%) were virtually quiescent, but the remaining 3 (12%) had almost the same channel activity, as exemplified in Fig. 7A. The channel activity mainly depended on the 70-pS channel, as can be seen from a current amplitude histogram in Fig. 7B that revealed a major peak at 2.9 pA. An averaged value for the major peak on the three patches was 2.9 ± 0.1 pA, and calculated single channel conductance was 71.6 ± 2.7 pS (n= 3). In any of these patches, no evidence for the 120-pS channel activity was found, although the 20-pS channel was seen open on occasion (see asterisks in Fig. 7Aa). Open-time histograms for the 70-pS channel were fitted with the sum of three exponential components (Fig. 7C), and averaged Oτ values for the respective fitted components were 0.55 ± 0.16 ms (relative proportion: 0.35 ± 0.04), 3.1 ± 0.7 ms (0.36 ± 0.33) and 17.4 ± 2.6 ms (0.29 ± 0.02) (n= 3). The latter two mean values differed significantly from the corresponding value for the 70-pS channel in M2-KO patches (0.55 ± 0.12 ms). As can be seen from Fig. 7Aa, long channel openings were each interrupted by brief shuttings (< ∼0.5 ms). The longest open durations measured in the different patches varied from 75.6 to 121.8 ms with a mean of 91.1 ± 15.3 ms (n= 3). No double opening of the channel was seen. The Po values varied from 0.03 to 0.05 with a mean of 0.042 ± 0.005 (n= 3). It should be noted that application of atropine (3 μm) in these active patches did not completely block channel activity but did profoundly reduce the frequency of openings and their duration, so that brief openings continued to occur at a lowered frequency (Fig. 7A). These data suggest that the M2 pathway that is functional in M3-KO patches targets the 70-pS but not the 120-pS channel. Additionally, the M2 and M3 pathways seem to differ in their effect on 70-pS channel gating.

In WT cells, 25 out of 40 patches (63%) were virtually quiescent, but the remaining 15 (37%) clearly active. These active patches could be tentatively classified into four groups based on different profiles of channel activity that they displayed. Figure 8A shows a typical example of recordings from a first group of four patches (10% of the total), in which a short segment of recording is illustrated at a high time resolution. The channel activity in this group was characterized both by long openings of the 70-pS channel interrupted by brief shuttings and by no noticeable activity of the 120-pS channel. As shown in Fig. 9A, current amplitude histograms revealed a major peak at 3.1 pA, and an averaged peak value and unitary conductance estimated from four patches were 2.8 ± 0.2 pA and 71.2 ± 3.8 pS, respectively. The maximal open times in the different patches varied from 100.7 to 803.2 ms with a mean of 376.5 ± 157.0 ms (n= 4). The Po was 0.14 ± 0.04 on average with patch-to-patch variations from 0.01 to 0.23 (n= 4), significantly greater than that for the 70-pS channel observed in M2-KO (0.08) or M3-KO patches (0.04). Open time analysis was carried out in three patches, because in the remaining one patch, long openings (> 100 ms) of channel events were seen, but the number of acquired events was too low for the fitting process. Figure 9B shows a histogram of apparent open-times (20 bins per decade) measured in the patch of Fig. 8A. The open-time histograms were well fitted by the sum of four exponential components; Oτ values (and relative proportions) of the four fitted components were 0.3 ms (0.27), 1.8 ms (0.30), 10.0 ms (0.29), and 97.6 ms (0.14). Similar fitting was also allowed in the other two patches, and the average Oτ values for the three patches for the four fitted components were 0.62 ± 0.17 ms (0.29 ± 0.08), 2.7 ± 0.5 ms (0.37 ± 0.06), 16.9 ± 4.1 ms (0.24 ± 0.03), and 121.1 ± 42.2 ms (0.10 ± 0.03).

A second group comprising three additional patches (7.5% of the total) was also characterized by long opening of the 70-pS channel with no notable 120-pS channel activity. However, open time histograms were not fitted with four components but rather well with three components (data not shown). The averaged Oτ values (relative proportions) for the respective components were 0.25 ± 0.03 ms (0.53 ± 0.04), 1.1 ± 0.1 ms (0.38 ± 0.07), and 13.2 ± 4.3 ms (0.08 ± 0.03) (n= 3 for each). The maximum open time and the Po value (43.6 ± 15.3 ms and 0.016 ± 0.005, n= 3) significantly differed from the respective corresponding values for the first group. The profiles of channel activity in this group resembled those seen in M3-KO patches.

A third group comprising six patches (15% of the total) had profiles similar to those characterized in M2-KO patches (cf. Figs 8B and 6A). Actually, the channel activity consisted of mixed gating of the 70-pS and 120-pS channels in brief open times, as judged from histograms for current amplitude (Fig. 9C) and apparent open-time (Fig. 9D). Based on the properties of five patches available for fitting analysis, the two major peaks of single channel current detected were 2.8 ± 0.1 and 4.9 ± 0.1 pA, corresponding to unitary conductances of 69.9 ± 1.2 and 121.8 ± 2.1 pS, respectively. The Po value was 0.04 ± 0.01 for the 70-pS and the 0.01 ± 0.001 for the 120-pS channel, and the respective Oτ values, determined with a single exponential function, 0.46 ± 0.11 and 0.32 ± 0.09 ms.

In the final group of the two remaining patches (5% of the total), as shown in Fig. 8C, channel activity was too complicated to be analysed. However, visual inspection of recording traces at a higher time resolution indicated mixed openings of the 70-pS, 120-pS and 20-pS channels (see a and b in Fig. 8C). Moreover, the 120-pS channel opened only briefly (a few milliseconds at the maximum), while longer openings of 10–100 ms occasionally occurred in the case of the 70-pS channel. From the channel activity of this group, it seemed as if M2-KO and M3-KO types of channel activity, as seen in M2-KO and M3-KO patches, were superimposed on each other.

The voltage dependency of 70-pS channel current was investigated in one patch of the first group by displacing membrane potential from the −40 mV holding potential between −80 mV and 40 mV, where channel activity at different potentials was observed for 10 s each (Fig. 10A). As shown in Fig. 10B, unitary current amplitude was a nearly linear function of the potential with a slope of 69.2 pS. In contrast, mean patch current (i.e. current integral that measures the transferred charge divided by the trace duration) showed U-shaped voltage dependence in negative potentials (Fig. 10C), like whole-cell mICat did in WT and M3-KO cells (see Fig. 3A and C). This finding supports the idea that the major part of whole-cell mICat in these cells depends on the 70-pS channel.

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Figure 10. I–V relationship of the 70-pS channel evoked by carbachol in the outside-out patches from WT cells A, carbachol (100 μm)-evoked 70-pS channel currents recorded at different holding potentials from the same outside-out patch. B and C, I–V relationship of the carbachol-evoked single channel current (B) and mean patch current (C) shown in A. The I–V relationship in B had a slope conductance of 69.2 pS. For measurement of mean patch currents in C, the transferred charge (current integral) at each holding potential was divided by the trace duration t.

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The channel activity observed in the WT patches was usually terminated after application of atropine (3 μm) or quinidine (10 μm) (see Fig. 8A and B), whereas in a few active patches, brief openings of the 70-pS channel still occurred at a low frequency in the presence of atropine, suggesting again that this channel has a constitutive gating activity. To confirm this, we tried to examine WT patches formed before carbachol application. In 3 out of 11 patches, a significant channel activity was seen, as exemplified in Fig. 11A. It consisted of brief openings of the 70-pS channel, as judged from an unitary current amplitude of 2.8 pA and a Oτ of 0.62 ms (Fig. 11B and C). When carbachol (10 μm) was applied to the spontaneously active patch, prolonged openings (up to 60 ms) of the 70-pS channel additionally occurred but ceased within 10–20 s (see Fig. 11Ab).

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Figure 11. The 70-pS channel is constitutively active A, typical example of single channel activity in the presence and absence of carbachol (100 μm) under the outside-out configuration from WT cells. Time-expanded, consecutive traces corresponding to a and b are shown in the lower panels. The asterisk indicates opening of the 20-pS channel. B, fitted levels amplitude (left hand panel) and open time (right hand panel) histograms for the 70-pS channel in the absence of carbachol shown in A. Gaussian function fitting to the amplitude histogram gave a peak amplitude of 2.8 pA. The open time distributions were fitted by the one exponential component with the time constant of 0.6 ms.

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The channel analysis in WT patches indicates that the 70-pS channel is the target for the M2/M3 pathway. Besides this pathway, the M2 and M3 pathways also exist in WT gut myocytes. Furthermore, the M2/M3 pathway can cause the 70-pS channel to open much longer than any other pathway.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

In the present study, using M2-KO, M3-KO, and M2/M3 double-KO mice as novel tools, we have attempted to identify muscarinic pathways leading to the activation of cationic channels and elucidate the underlying signal transduction mechanisms in intestinal smooth muscle cells.

Muscarinic pathways for cationic channel activation

Although M2 and M3 muscarinic receptors are preferentially expressed in gastrointestinal smooth muscle cells, a recent RT-PCR study suggests the possible expression of all five muscarinic receptor subtypes (M1–M5) in gastric myocytes (So et al. 2003). The present study has shown that only the M2 and M3 subtypes participate in cationic channel activation in intestinal myocytes. Carbachol evoked mICat in M2-KO or M3-KO cells but not in M2/M3 double-KO cells, although all mutant cells showed normal G protein-cationic channel coupling, as judged from their ability to generate cationic currents in response to GTPγS (Fig. 1). Similarly, M2 and M3 receptors are the only muscarinic receptors involved in muscarinic depolarization (see Fig. 5), as well as the contractile response to carbachol or cholinergic nerve stimulation in ileal smooth muscles (Unno et al. 2005, 2006a).

Measurements of IK-Ca and cyclic AMP responses to carbachol indicated that the M2 and M3 receptor subtypes can couple normally to their respective G protein/effector systems (i.e. the Gi/adenylyl cyclase and the Gq/PLCβ, respectively) in M3-KO and M2-KO cells, respectively. This observation suggests that the carbachol (100 μm)-evoked mICat in M2-KO or M3-KO cells represents the full response to stimulation of either M3 or M2 receptors. However, the M2- and M3-mediated mICat (13 and 24 pA, respectively) responses were much smaller than the WT value (209 pA), and the mICat evoked in WT cells is not a simple additive mixture of mICat responses observed with M2-KO or M3-KO cells. These observations suggest the existence of an additional pathway that requires the presence of both M2 and M3 receptors (M2/M3 pathway) contributing to mICat in intact gut myocytes. In guinea-pig ileum, mICat is competitively inhibited by M2-preferring antagonists and severely depressed by M3-preferring ones with no notable change in the agonist EC50 (Zholos & Bolton, 1997). It is also blocked or markedly reduced by PTX, anti-Gαo antibody or a PLC inhibitor (Inoue & Isenberg, 1990; Komori et al. 1992; Okamoto et al. 2002, 2004; Yan et al. 2003; Zholos et al. 2004a). Mouse intestinal mICat displayed similar features (Sakamoto et al. 2006). These pharmacological findings have led to the hypothesis that M2 and M3 receptors synergistically activate mICat in gut smooth muscles. The present study using mutant mice provides more direct evidence for the existence of this synergistic M2/M3 pathway.

Target channels for the muscarinic pathways

To specify the cationic channels that the individual muscarinic pathways target, we observed channel activity in outside-out patches excised from M2-KO, M3-KO or WT cells after their exposure to carbachol, as described by Zholos et al. (2004b). The channel analysis made in symmetrical Cs+ solutions with [Ca2+]i≈ 100 nm revealed three types of muscarinic cationic channel with different unitary conductances of 20, 70 and 120 pS. A substantial similarity was found between our results and those obtained in guinea-pig or murine ileal myocytes (Zholos et al. 2004b; Dresviannikov et al. 2006: see Introduction). Our major novel finding is that the M2 or the M2/M3 pathway targets 70-pS channels and that the M3 pathway tergets both 70-pS and 120-pS channels. In active M2-KO patches (M3 pathway), 70-pS and 120-pS channel activities were usually observed together, but the former activity was predominant in the number of open events and Po, suggesting that the 70-pS channel is the major contributor to mICat in M2-KO cells. If so, the M2-KO mICat should have shown a U-shaped I–V relationship, like mICat in M3-KO cells did, because of the dependence on the 70-pS channel. However, it rather showed a linear I–V relationship (Fig. 3). Zholos et al. (2004b) found in guinea-pig ileal myocytes that the mean patch current of the 130-pS channel (corresponding to our 120-pS channel) is almost a linear function of membrane potential. Accordingly, the mICat in M2-KO cells is more likely to be due to 120-pS than to 70-pS channel activity. The guinea-pig 130-pS channel is suggested to have diffusible second messengers involved in its activation, since the channel activity was rarely observed in outside-out patches compared with cases for cell-attached patches (Zholos et al. 2004b). Such circumstances may account for the apparent contradiction between the outside-out and whole-cell data with M2-KO cells. In this context, we also examined the effects of OAG, an analogue of the diffusible second messenger DAG formed via the M3–Gq–PLCβ system. We found that OAG (30 μm) produced a sustained cationic current in WT cells (25.6 ± 6.4 pA; n= 7) and that the I–V curve for the OAG current was roughly linear. This current was also insensitive to voltage-gated Ca2+ influxes. Also, in a WT outside-out patch a significant 120-pS channels activity occurred in response to OAG (T. Sakamoto, T. Unno & S. Komori, unpublished observation). Similar OAG-induced whole-cell currents are found in guinea-pig ileal myocytes (Okamoto et al. 2004).

Active patches from WT cells could be classified into four groups, based on the different features of gating activity of the 70-pS and 120-pS channels (Figs 8 and 9). The view that the 70-pS channel is the target of the M2/M3 pathway is strongly supported by the observation that 27% of the active patches displayed a unique gating activity of 70-pS channels that M2-KO or M3-KO patches never displayed. The 70-pS channel activity was characterized by a sequence of four open states including a considerably long one (see the next section), but also by the lack of 120-pS channel activity. The mean patch current due to the unique channel activity showed a U-shaped I–V relationship as did WT or M3-KO whole-cell mICat, which is consistent with the above view, because WT mICat is predicted to arise mainly through the M2/M3 pathway and M3-KO mICat is thought to be mediated by the 70-pS channel.

About 13% of WT patches (the sum of 3 patches resembling M3-KO patches and 2 patches exhibiting mixed type of M2-KO and M3-KO patches: see Results) showed M2-type channel activity, and ∼20% (the sum of 6 patches resembling M2-KO patches and 2 patches of the mixed type patches) displayed M3-type channel activity. Interestingly, these relative percentages were near the corresponding values for M3-KO (12%) and M2-KO patches (15%), respectively. It is thus likely that the M2 and M3 pathways are functioning independently in WT cells.

Functional units and signal transduction mechanisms

Previous extensive whole-cell studies in guinea-pig and mouse gut myocytes have hypothesized that synergistic interaction of an M2–Go system and an M3–PLC system plays a crucial role in mICat generation without significant participation of IP3, DAG, or Ca2+-store depletion (Zholos & Bolton, 1997; Yan et al. 2003; Zholos et al. 2004a; Okamoto et al. 2004; Sakamoto et al. 2006). In this context, the three muscarinic pathways identified in the present study can be described as depicted in Fig. 12.

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Figure 12. Scheme depicting the predicted three distinct muscarinic signalling pathways mediating the activation of cationic channels in gut smooth muscle cells The M3 pathway, via the M3–Gq–PLCβ system, leads to activation of the 70-pS and 120-pS cationic channels in brief opening mode and concurrently to IP3-induced Ca2+ release. The 70-pS and 120-pS cationic channels are activated probably through loss of PIP2 contents and DAG formation, respectively. The M2 pathway transduces signals from M2 receptors via Go to the 70-pS cationic channel and shifts the brief open mode toward a longer open mode. The M2/M3 pathway transmits M2 signals via Go and M3 signals via a PLC, independent of Gq, to the 70-pS cationic channel, resulting in a much longer open mode; this pathway does not work well when either the M2 or M3 receptor is lacking, or either Go or PLC is inactivated. Our results support the concept that M2 and M3 receptors are part of a higher molecular order signalling complex, analogous to the signalling complex mediating photo-signal transduction including TRP channels in Drosophila (Clapham et al. 2001; Minke & Cook, 2002). All three pathways are thought to induce excitatory membrane potential responses. The M2/M3 pathway, but not the M2 or M3 pathway, involves processes in which Ca2+ has a potentiating effect on channel activation, suggesting that the M3 pathway may facilitate the function of the M2/M3 pathway through IP3-induced Ca2+ release. See text for details.

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The M3 pathway This pathway consists of the well-known M3–Gq–PLCβ system and both 70-pS and 120-pS channels. As mentioned earlier, the 120-pS channel may be activated by the second messenger DAG from PLCβ hydrolysis of PIP2. DAG-dependent mechanisms for cationic channel activation have also been suggested in gastric and vascular myocytes (Lee et al. 2003; Albert & Large, 2006). The activation mechanism of the 70-pS channel is probably different. In the present outside-out patch experiments, 70-pS channel activity was considerably more prominent as compared with the 120-pS channel, suggesting that 70-pS channel activation involves membrane-delimited mechanisms rather than diffusible second messengers. Recent evidence suggests that a certain type of TRP channel, TRPV1, is under inhibitory regulation by PIP2, and loss of PIP2 from the membrane relieves the channel from inhibition, leading to channel opening (Chuang et al. 2001). Since muscarinic cationic channels in gut myocytes are thought to belong to the TRP channel family (Walker et al. 2001; Lee et al. 2003), it seems likely that the 70-pS channel activation is caused by loss of PIP2 due to PLCβ hydrolysis; as a result, both 70-pS and 120-pS channel activities were often seen in an active M2-KO patch. Our previous observation that the PLC inhibitor U73122 almost completely blocked whole-cell mICat (Okamoto et al. 2004) is consistent with the possible importance of PIP2 metabolism for muscarinic cationic channel opening (see below). However, it remains unclear why both channels usually coexist in one active patch.

The M2 pathway This pathway includes the M2-Go system and the 70-pS channel. The 70-pS channel activity mediated by M2 receptors was clearly different from that mediated by M3 receptors (cf. Figs 6 and 7). The M3-mediated activity consisted of brief openings with a single open state (Oτ= 0.6 ms) and almost disappeared upon addition of atropine, whereas the M2-mediated activity had a series of three open states (Oτ= 0.6, 3.0 and 17.4 ms) and was not completely blocked by atropine; brief openings continued to occur in the presence of atropine, probably due to a spontaneous gating activity of the channel. The spontaneous 70-pS channel activity demonstrated in WT patches without carbachol stimulation had a brief open state (Oτ= 0.6 ms) similar to that observed in M2-KO patches. In rabbit ear artery myocytes, TRP-like cation channels are constitutively active and their openings are suggested to be regulated by spontaneous activation of the Gq–PLC pathway (Albert & Large, 2004). Thus, in M3-KO patches, it is possible that a PLC is activated by spontaneous action of G proteins or by other intrinsic mechanisms coupled with receptors other than M3 subtype, and loss of membrane PIP2 may occur, which is sufficient for the 70-pS channel to be gated. Taken together, it is plausible that the M2-Go system does not directly open the channel, but rather requires loss of PIP2 to permit channel gating so that the M2-Go system can shift a brief open state (Oτ= 0.6 ms) toward longer open states (Oτ= 3.0 and 17.4 ms).

The M2/M3 pathway This pathway consists of the M2-Go and M3-PLC systems including the 70-pS channel. The M3-PLC system seems virtually independent of Gq/11 proteins, because without influences by intracellular Ca2+ release, mICat was not significantly affected by anti-Gαq/11 antibody (Yan et al. 2003; Unno et al. 2006b). Thus, the PLC included in this complex may not be identical to that coupling to Gq/11 proteins (PLCβ). A striking feature of the M2/M3 pathway is to trigger a much longer open state, compared with the M2 pathway, so that 70-pS channel openings can occur as a series of four open states of Oτ= 0.6, 2.7, 16.9 and 121.1 ms (Fig. 9B), providing a substantial rise in Po and therefore playing the major role in mICat generation. This potent effect is most likely dependent on the M3-PLC system, which is predicted to decrease PIP2 levels so that the channel can be freed enough to open for long durations in response to concurrent signals from the M2-Go system. The concept of M2/M3-mediated channel activation is consistent with the prediction of Zholos et al. (2004b) that the effect of muscarinic receptor stimulation on the guinea-pig ileal 60-pS channel (corresponding to our 70-pS channel) is to increase the probability of channel transition towards pairs of longer open/shorter closed states (see their Scheme I for channel gating mode). Based on their four open/closed states model, constitutive activity of the 70-pS channel may represent the lowest pair state with long closings and very short openings (state 1), and receptor activation may move the channel progressively towards the extreme fourth pair state with long openings and short closings. M3 receptor activation may increase the open probability of the 70-pS channel in pair state 1 and be weakly active in moving it out of pair state 1. However, M2 receptor activation may be very effective in moving the channel from pair state 1 towards pair states 2, 3 and 4, and the combination of M2 and M3 stimulation even more effective, thus increasing mean open times in the WT patches. Mean open times would depend on the mixture of pair states which change progressively depending on the strength of activation of the receptor and G protein.

The M2/M3 pathway has a built-in feature through which Ca2+ can potentiate channel activation. No such feature exists for the other two pathways, suggesting the possibility that the M3–PLC system is important for the observed Ca2+-sensitivity. Little known is about how M3 receptors and PLC are functionally linked and whether the PLC activity is sensitive to Ca2+. Another possibility is that there is a Ca2+-sensitive step somewhere in a sequential process through which the M2–Go system modifies channel gating mode under the cooperation of the M3–PLC system. Further studies are needed to clarify this issue.

In equine tracheal myocytes, an M2 and M3 synergistic mechanism has also been suggested for mICat generation, but the role of M3 is to evoke a rise in [Ca2+]i such that caffeine, a Ca2+ releaser, could mimic the effects of M3 receptor activation (Wang et al. 1997). However, in intestinal myocytes, the activation of both receptors per se is obligatory for current generation. Therefore, the synergism of M2 and M3 receptors may differ between the two kinds of myocyte.

In the Drosophila phototransduction cascade, signalling depends on the TRP channel, G protein, PLC and others which are all organized into supra-molecular complexes by certain scaffolding proteins (Minke & Cook, 2002). By analogy, the M2/M3 functional units might exist as complexes formed by M2 and M3 receptors together with Go protein, atypical PLC, the 70-pS channel and other molecules. Co-expression studies of M2- and M3-encoding genes have suggested the possible formation of M2/M3 heterodimers endowed with novel pharmacological properties (Maggio et al. 1999).

Physiological significances of the three muscarinic pathways

The primary role of the muscarinic cationic channel is to depolarize the membrane, so admitting Ca2+ into the cell via voltage-gated Ca2+ channels (Bolton, 1979; Unno et al. 2003). The present whole-cell current-clamp experiments revealed that the M2 and M3 pathways are effective enough to produce a significant depolarization (Fig. 5). Combined with the results from channel analysis, the M2-mediated depolarization is attributable to 70-pS channel activation. On the other hand, the M3-mediated depolarization seems to be preferentially due to the activity of the 120-pS channel, as discussed above. The maximum sizes of depolarization were ∼10 mV and ∼40 mV for the M2- and the M3-mediated pathway, respectively (Fig. 5), but the difference between both sizes cannot simply be explained by that between the mICat sizes (13 and 24 pA). One possibility is that the M3-mediated depolarization involves a contribution from Ca2+-activated Cl channels, since the M3 pathway causes the activation of these channels via IP3-induced Ca2+ release (Sakamoto et al. 2006). It is also possible that the 120-pS channel has a greater permeability ratio of Na+/Cs+ than the 70-pS channel, whereby the former channel may mediate an even greater inward current in a Na+-rich solution in which membrane potential changes were measured.

Previously, we used gut muscle strips from the same mutant mouse strains as used for the present study to characterize M2- and M3-mediated contractions (Unno et al. 2005). The M2 contraction totally depends on Ca2+ entry associated with depolarization and is sensitive to pertussis toxin (PTX), known to disrupt functional links between M2 and Gi/Go proteins, consistent with the M2 pathway depicted in Fig. 12. The M3 contraction is resistant to PTX and depends on multiple mechanisms, all of which elevate [Ca2+]i, including both voltage-dependent and -independent Ca2+ entries and intracellular Ca2+ release. These features agree well with those predicted from the M3 pathway (Fig. 12). There is evidence that the 60- or 70-pS channel, the major contributor to mICat, shows very poor Ca2+ permeability (Pacaud & Bolton, 1991; Kim et al. 1998), suggesting that these channels are not involved in voltage-independent Ca2+ entry. Therefore, the 120-pS channel seems likely to participate in voltage-independent as well as voltage-dependent Ca2+ entry. However, further work is needed to test this possibility.

The carbachol-evoked depolarization observed in WT cells may arise through the three muscarinic pathways. The depolarizing activity of the M2/M3 pathway is predicted to be strengthened by the M3 pathway through the IP3-induced Ca2+ release, for reasons mentioned earlier. In fact, Ca2+ store depletion significantly reduces carbachol-evoked depolarization in guinea-pig ileal myocytes (Unno et al. 2000). Such interaction between these two pathways may account at least partly for the fact that a small fractional occupancy of muscarinic receptors (∼10%) is sufficient to evoke a full depolarization (Bolton, 1972; Unno et al. 2000, 2003).

Conclusion

In summary, we have demonstrated that mouse gut smooth muscle cells are endowed with three distinct pathways mediating mICat generation. Two of these pathways are initiated by activation of either of M2 and M3 receptors and target 70-pS channels or both 70-pS and 120-pS channels, respectively. The third pathway requires the presence of both M2 and M3 receptors to be effective and targets 70-pS channels. This M2/M3 pathway most strongly activates the 70-pS channel resulting in a considerably longer open state and a substantial rise in the opening probability. Thus, the M2/M3 pathway is the major mediator of whole-cell mICat and most potently depolarizes the membrane. The delineation of the M2/M3 pathway is consistent with the existence of a signalling complex involving the M2–Go system, the M3–PLC system and cationic channels predicted to mediate mICat. These findings provide novel insights into the signal transduction mechanisms underlying the muscarinic regulation of electrical and mechanical activity of intestinal smooth muscle. Further studies are needed to identify the molecular basis of the muscarinic cationic channels and to elucidate their activation mechanisms in more detail.

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  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

We thank Ms Mai Uchiyama for excellent technical assistance. This work was supported by a Grant-in-Aid Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 16380199 and 17580253).