Involvement of Ca²⁺ in Signaling Mechanisms Mediating Muscarinic Inhibition of M Currents in Sympathetic Neurons

Abstract

Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M₁ muscarinic receptors coupled to the G_q protein. Although PIP₂ depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (I_M), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of I_M with Ca²⁺ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of I_M was not blocked either by PIP₂ or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of I_M uses multiple signaling pathways by blocking both PIP₂ depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of I_M. Additionally, muscarinic inhibition of I_M was not prevented either by sequestering of G-protein βγ subunits from G_α-transducin or anti-G_βγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca²⁺ signals in muscarinic inhibition of I_M. Ca²⁺ measurements showed that muscarinic stimulation increased intracellular Ca²⁺ and was comparable to the Ca²⁺ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of I_M. In contrast, muscarinic inhibition of I_M was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca²⁺ signaling in muscarinic modulation of I_M. The differential effects of EGTA and BAPTA imply that Ca²⁺ microdomains or spatially local Ca²⁺ signals contribute to inhibition of I_M.

Introduction

Neural M-type (KCNQ/Kv7) K⁺ channels are low threshold voltage-gated K⁺ channels that play a crucial role in regulating excitability of neurons (Delmas and Brown 2005; Cooper and Jan 2003). Breakdown of M channels by loss of function mutations or pharmacological inhibitors leads to neuronal hyperexcitability (Schroeder et al. 1998; Delmas and Brown 2005). Indeed, linopirdine, an M channel blocker, has acute cognition-enhancing properties in some animal experiments (Aiken et al. 1996), whereas M channel openers, such as retigabine, are currently being developed as novel antiepileptic drugs (Cooper and Jan 2003).

M currents (I_M) were originally described in sympathetic neurons (Brown and Adams 1980; Constanti and Brown 1981) and named due to their inhibition by muscarinic acetylcholine receptors. However, the signal transduction mechanism underlying muscarinic inhibition of I_M remains uncertain. Despite the fact that muscarinic inhibition of I_M was shown to be mediated by G_q-protein activation (Caulfield et al. 1994; Haley et al. 2000), typical downstream pathways of G_q such as PKC or IP₃-mediated Ca²⁺ signals were not involved in muscarinic inhibition of I_M (Beech et al. 1991; Bosma and Hille 1989; Cruzblanca et al. 1998; del Rio et al. 1999). It was shown that neuronal I_M is encoded by KCNQ family genes (Wang et al. 1998) and activities of KCNQ channels depend on phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasma membrane (Li et al. 2005; Suh et al. 2006; Zhang et al. 2003; Kim et al. 2016). Recent progress elucidated that PIP₂ modulate KCNQ2/KCNQ3 channel opening by interacting synergistically with a minimum of four cytoplasmic domains (Hernandez et al. 2008a; Choveau et al. 2018). These led to a hypothesis that depletion of PIP₂ by muscarinic stimulation is responsible for inhibition of I_M (Delmas and Brown 2005; Suh and Hille 2002).

I_M is negatively modulated by other G_q-coupled neurotransmitter receptors, such as bradykinin B₂ receptors, and purinergic P2Y receptors (Bofill-Cardona et al. 2000; Jones et al. 1995). The mechanism involved in bradykinin-induced inhibition of I_M has been particularly well characterized, and it has been shown that calmodulin activation induced by IP₃-mediated intracellular Ca²⁺ signals is responsible for I_M inhibition (Gamper et al. 2005; Gamper and Shapiro 2003). At present, it appears to be generally accepted that I_M is regulated by three modulatory pathways: PIP₂ depletion for muscarinic inhibition and second messengers, such as Ca²⁺/CaM for bradykinin-induced inhibition (Hernandez et al. 2008b), protein kinase C (Lee et al. 2010; Kosenko et al. 2012), and post-translational modification (Kim et al. 2016; Qi et al. 2014). However, despite many studies investigating the PIP₂ depletion hypothesis for the mechanism of muscarinic inhibition of I_M (Ford et al. 2003; Suh and Hille 2002; Suh et al. 2006; Winks et al. 2005; Zhang et al. 2003), direct evidence is still lacking. In addition, since PIP₂ mobility determines the spatial and temporal profiles of PIP₂ depletion, the idea that PIP₂ depletion acts as the M₁ muscarinic signal in neurons requires that PIP₂ diffusion be slow (Cho et al. 2005). Yet, in general, PIP₂ diffuses rapidly in neurons (van Rheenen and Jalink 2002; Cho et al. 2005), making this hypothesis unlikely. The PIP₂ depletion hypothesis was supported by a result demonstrating that increasing PIP₂ concentration of sympathetic neurons by overexpressing the synthetic enzyme phosphoinositide-5 (PI-5)-kinase can block muscarinic inhibition of I_M. Kruse and Whitten recently developed a model that describes altering M₁R surface density and PI-5-kinase activity regulating the excitability of rat SCG neurons (Kruse and Whitten 2021). However, it is not certain whether increased resting PIP₂ levels caused by overexpression of PI-5-kinase can inhibit PIP₂ depletion induced by receptor-mediated activation of PLC (Winks et al. 2005). Given that PIP₂ can modulate a variety of cellular processes, including cortical actin organization, membrane ruffling, vesicle trafficking, and gene expression (Di Paolo and De Camilli 2006), the possibility that normal signaling mechanisms involved in muscarinic inhibition of I_M are altered in excess PIP₂ cannot be excluded. To prove whether receptor-mediated inhibition is mediated by PIP₂ depletion, it is important to demonstrate that the inhibition is rescued by applying normal concentrations of exogenous PIP₂. Indeed, inhibition of GIRK channels by phenylephrine, endothelin, and PGF2α in cardiac myocytes, which were shown to be mediated via PIP₂ depletion, are completely abolished by internally perfused 10-μM PIP₂ (Cho et al. 2005, 2001). However, muscarinic inhibition of KCNQ channels was only partially reduced even by 500-μM PIP₂ (Robbins et al. 2006), which can be considered inconsistent with the PIP₂ depletion hypothesis. In the present study, we investigated the mechanism involved in muscarinic inhibition of I_M by examining various possible signaling components in rat superior cervical ganglion (SCG) sympathetic neurons and HEK cells expressing KCNQ channels.

Materials and Methods

Ethics Approval

This study was reviewed and carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) at Sungkyunkwan University School of Medicine (SUSM). SUSM is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility and abides by the Institute of Laboratory Animal Resources Guide. The animals were maintained in standard environmental conditions (25 ± 2 °C; 12/12-h dark/light cycle), were given ad libitum access to water and food, and were housed under veterinary supervision at the Laboratory Animal Research Center, SUSM. Sprague–Dawley rats were purchased from Orient Bio Inc. (Sungnam, South Korea). The authors understand the ethical principles under which The Journal of Physiology operates and the experiments comply with the animal ethics checklist described in Grundy (Grundy 2015).

Cell Culture, Transfection, and SCG Isolation

SCG neurons were cultured from 3- to 4-week-old male rats as previously described (Gamper et al. 2004). HEK293 cells were handled as previously described (Cho et al. 2006). Transfections were made using Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA) and green fluorescent protein (GFP) was used as a reporter. Plasmids encoding human KCNQ2 (GenBank accession number AF110020) and rat KCNQ3 (GenBank accession number AF091247) were kindly provided by Mark Shapiro (University of Texas Health Science Center, San Antonio, TX).

Electrophysiological Recordings

Current measurements were made with the whole-cell patch-clamp technique. Voltage clamp was performed with an EPC-8 amplifier (HEKA Instruments, Lambrecht, Germany). Filtered signals (1–2 kHz) from a patch-clamp amplifier were fed into an AD/DA converter (PCI-MIO-16E-4, National Instruments, Austin, TX), digitized at 5 kHz and stored digitally in later analysis. Electrodes were pulled from borosilicate capillaries (World Precision Instruments, Inc., Sarasota, FL) using a pipette puller (PP-83, Narishige, Tokyo) and positioned precisely with a micromanipulator (MP 225, Sutter Instrument Company, Novato, CA). The pipettes had a resistance of 2–3 MΩ when filled with pipette solution. Data were not corrected for liquid junction potential (−9 mV). For dialysis, we waited > 5 min before starting the experiment. The perfusion system was a homemade 100-µl perfusion chamber through which solution flowed continuously at 5 ml/min. All recordings were carried out at room temperature (22–24 °C).

The normal external solution for HEK293 cell and rat SCG neuron recording was as follows (in mM): 143 NaCl, 5.4 KCl, 5 HEPES, 0.5 NaH₂PO₄, 11.1 glucose, 0.5 MgCl₂, 1.8 CaCl₂, and pH 7.4 adjusted with NaOH. The pipette solution was as follows (in mM): 126 KMeSO₄, 14 KCl, 10 HEPES, and 3 MgCl₂ (pH 7.24 adjusted with KOH). 500 nM of TTX (Tocris, St. Louis, MO, Cat. #1069) and 0.1 μM of CdCl₂ were included in external solution to block Na⁺ and Ca²⁺ currents.

[Ca²⁺]_i Measurement

[Ca²⁺]_I, the concentration of intracellular free Ca²⁺, was measured by loading cells with 2-μM Fura 2-acetoxymethyl ester (Fura 2-AM) (Molecular Probes, Eugene, OR) for 30 min in the recording medium. Changes in Ca²⁺ were estimated from the ratio of Fura 2 fluorescence (at 500 nm) with alternate excitation at wavelength of 340 and 380 nm, generated by a Lambda DG-4 monochromator (Sutter Instrument Company). Fura 2 ratio data (F340/380), [Ca²⁺]_i, were measured at a frequency of 2 Hz. Intensity of illumination was minimized to reduce bleaching of Fura 2, while maintaining adequate levels of signal. Fura 2 images for digital analysis were generated using a system based on an Olympus IX71 microscope coupled to an image intensifier and CCD camera and were analyzed with MetaFluor software (MDS Analytical Technologies, Sunnyvale, CA).

Statistical Analysis

Results in the text and the figures are presented as mean ± S.E.M. (n = number of cells tested). Statistical analyses were performed using Student’s t test. P‐values of < 0.05 were considered statistically significant.

Results

Characterization of I_M and Muscarinic Modulation of M Channels in SCG Neurons

I_M was originally characterized as outward K⁺ currents suppressed by muscarinic stimulation (Brown and Adams 1980) and specific blockers to inhibit I_M, such as linoperdine and XE991, were developed later (Aiken et al. 1996; Cooper and Jan 2003). To investigate the mechanism of muscarinic modulation of I_M in rat SCG neurons, we first compared the current component inhibited by oxotremorine-M (oxo-M), a muscarinic agonist, and that inhibited by XE991. Here we used the whole-cell patch-clamp mode, a method suitable for preserving intracellular microdomains, investigating localized signaling, and directly modulating the signaling molecules driving muscarinic inhibition of M channels. To prevent the potential “run-down” of I_M in the whole-cell configuration, after the pipette ruptured the membrane, we allowed I_M to stabilize before tracking muscarinic inhibition. Step depolarizing pulses were applied in 10-mV steps from a holding potential of −60 mV. In Fig. 1A, representative current traces obtained in the control condition and after applying oxo-M (10 μM) or XE991 (50 μM) are shown. We obtained the current component inhibited by oxo-M (oxo-M-sensitive) and that inhibited by XE991 (XE991 sensitive) by subtracting current traces in the presence of oxo-M or XE991, respectively, from the control current traces (Fig. 1B, left panel). In Fig. 1B (right), current–voltage (I-V) relationships for total currents in control (squares), oxo-M-sensitive currents (closed circles), and XE-991-sensitive currents (open circles) were plotted. The plot shows that oxo-M-sensitive currents and XE-991-sensitive currents are similar over the voltage range tested between −60 mV and + 20 mV. Assuming that XE-991-sensitive currents represent I_M, I_M comprises 26.49 ± 1.41% of total outward currents at + 20 mV and 10-μM oxo-M inhibits I_M by 79.90 ± 13.62% (at + 20 mV, n = 7).

Fig. 1

Isolation of I_M from SCG neurons. A Families of current elicited by voltage steps from −60 mV to + 20 mV, in 10-mV intervals, before (left panel) and after application of 10-μM oxo-M (middle) or 50-μM XE991 (right). Holding potential −60 mV. Inset shows the pulse protocol. B left, oxo-M-sensitive and XE991-sensitive currents; right, steady-state current–voltage (I–V) relationships for oxo-M-sensitive and XE991-sensitive currents shown in left. C left, superimposed deactivation tails for control (black), oxo-M sensitive (red), and XE991-sensitive currents (green) by hyperpolarizing steps to −60 mV from −20 mV (upper panel) or + 20 mV (lower panel); right, I–V relationships for control, oxo-M-sensitive, and XE991-sensitive tail currents

In Fig. 1C, we compared deactivation tail currents (I_tail) recorded upon repolarization (indicated as square boxes in Figs. 1A and B) and plotted the amplitude of I_tail against step depolarization voltage. To avoid contamination of capacitive currents and fast components that may not be attributable to I_M, we measured the amplitude of I_tail by measuring the average current level between 30 and 50 ms after repolarization and subtracting the current level at steady state. The result shows the similarity between I_tail of oxo-M-sensitive currents and XE-991-sensitive currents. Furthermore, at voltage ranges up to −20 mV, I_tail of oxo-M-sensitive currents and XE-991-sensitive currents are almost equivalent to I_tail of control currents, indicating that I_tail corresponding to −20-mV depolarization is mostly attributable to I_M. In the present study, therefore, we regarded I_tail as an indication of I_M.

The concentration-dependent effects of oxo-M to inhibit I_M were tested, while changes of I_tail were monitored in 5-s intervals by applying a hyperpolarizing pulse to −60 (or −55) mV for 1.5 s from the holding potential of -20 mV. Extent of inhibition induced by 1-μM and 10-μM oxo-M were 42.54 ± 3.51% (n = 12) and 84.13 ± 7.34% (n = 16), respectively (Fig. 2C).

Fig. 2

Muscarinic inhibition of I_M was not blocked either by supply of PIP₂ or by PKC inhibitor. The current amplitudes were measured as the deactivation tail current induced by 1-s hyperpolarizing steps to −60 mV from a holding potential of −20 mV at 5-s intervals. A The effects of 1-µM oxo-M (left) or 10-μM oxo-M (right) on I_M in cells which were patched with 20-μM diC8-PIP₂. B Time course of I_M amplitude during cumulatively increasing concentration of oxo-M indicated. Neurons were pretreated by 1-µM calphostin C without (left) and with (right) 20-µM diC8-PIP₂ in the patch pipet. Inset shows the pulse protocol and representative current traces of left panel. C Summary of the percent inhibitions of I_M by oxo-M in control conditions or in cells applied with various concentrations of PIP₂ or calphostin C. Values are expressed as means ± S.E.M. NS, not significant (p > 0.05)

Re-examination of the Current Hypothesis: PIP₂ Depletion and PKC Activation

PIP₂ depletion and PKC activation have been considered to be the most likely candidates involved in muscarinic inhibition of I_M (Delmas and Brown 2005). However, previous studies have found that muscarinic inhibition of I_M was not fully blocked by application of exogenous PIP₂ or PKC inhibitors (Brown and Yu 2000; Ford et al. 2003; Hoshi et al. 2003; Robbins et al. 2006; Shapiro et al. 2000). We confirmed that exogenous PIP₂ (up to 200 μM) had little effect on muscarinic inhibition of I_M (Fig. 2A). Consistent with the data from sympathetic neurons, in HEK293 cells expressing KCNQ2/KCNQ3 channels and M1 muscarinic receptors, application of PIP₂ did not block the effect of oxo-M on I_M (Yoon 2010). To apply PIP₂ into cells, diC8-PIP₂ was added to the pipette solution, and the conventional whole-cell mode was accomplished by rupturing membranes after a giga-seal was made. We have recently shown that application of diC8-PIP₂ disrupts M channel regulation induced by the altered channel’s affinity for PIP₂ (Lee et al. 2010), suggesting that exogenous PIP₂ is effectively delivered to the plasma membrane and available to M channels. We also confirmed that calphostin C, a highly specific PKC inhibitor, did not affect muscarinic inhibition of I_M (Fig. 2B). Summarized data are shown in Fig. 2C. In addition, a higher dose of calphostin C (up to 10 μM) and two other PKC inhibitors, 1-μM chelerythrine and 100-nM bisindolylmaleimide I, were tested and neither affected oxo-M-induced M current modulation (SI 1).

We then hypothesized that muscarinic inhibition of I_M uses multiple signaling pathways such that inhibition can only be blocked when multiple pathways are simultaneously suppressed. To test this hypothesis, both PIP₂ depletion and PKC activation were prevented with exogenous PIP₂ (20 μM) and calphostin C (1 μM). In the presence of 20-μM PIP₂ and calphostin C, however, 10-μM oxo-M was still capable of inhibiting I_M (Fig. 2B and C).

Looking for New Possibilities

Since we did not find evidence that messengers downstream of the Gα_q pathway mediate muscarinic inhibition of I_M, we searched for other possibilities. It is well known that the G_βγ dimer mediates voltage-dependent modulation of N-type Ca²⁺ channels through numerous neurotransmitters, including noradrenaline, acetylcholine, and dynorphin (Herlitze et al. 1996; Ikeda 1996; Tsien et al. 1988). We tested the involvement of the G_βγ subunit in oxo-M-induced I_M modulation using HEK cells transfected with G_α-transducin (G_αTD) together with KCNQ2, KCNQ3, and M₁ muscarinic receptors. G_αTD is a G_α subunit which predominantly attains the GDP-bound state when overexpressed in SCG neurons and buffers G_βγ (Kammermeier and Ikeda 1999). As shown in Fig. 3A, expression of G_αTD did not affect muscarinic inhibition of the KCNQ current by 1-μM oxo-M (87.84 ± 4.02% (n= 4) in controls or 89.77 ± 7.74% (n = 5) in G_αTD-expressed cells, p > 0.05). In addition, scavenging G_βγ subunits with intracellular anti-G_βγ antibody in SCG neurons was also tested. In the presence of anti-G_βγ antibody, 10-μM oxo-M-induced inhibition of M current was not different from control conditions. (Fig. 3B). These data implicate that G_βγ subunits do not have a critical role in muscarinic inhibition of I_M.

Fig. 3

G_βγ-buffering proteins did not affect muscarinic modulation of I_M. A Left, time course of I_M amplitude during cumulatively increasing concentrations of oxo-M in control HEK cells expressing KCNQ2/3 channels and M₁ muscarinic receptors (upper panel) and cells expressing channels and M1Rs plus Gα-transducin (lower panel). Right, summary of the percent inhibitions of I_M by oxo-M, collected as in left. B Left, time course of I_M in SCG neurons loaded with anti-G_βγ antibody. Right, summarized data for the effects of anti-G_βγ antibody on muscarinic inhibition of I_M. Values are expressed as means ± S.E.M

Recently, it was reported that receptor activation regulates channel activity by affecting the surface expression of channel proteins (Chung et al. 2009). Because KCNQ channels are known to be targets of trafficking, i.e., insertion into and removal from the plasma membrane (Etxeberria et al. 2008; Schuetz et al. 2008), we tested the possibility that KCNQ channel trafficking is involved in muscarinic inhibition of I_M. However, a block of protein trafficking with 50-μM blebbistatin, a cell-permeable inhibitor of class-II myosins, did not affect muscarinic inhibition of I_M (SI 2). In addition, pretreatment of a protein trafficking inhibitory cocktail including chloroquin (100 μM), monensin (100 μM), and nocodazole (20 μM) had no effect on muscarinic modulation of I_M (data not shown), suggesting that KCNQ channel trafficking did not contribute to the regulation of I_M by M₁ muscarinic receptors.

Revisiting Ca²⁺-Dependent Hypothesis

Recently, the role of Ca²⁺ signaling in I_M inhibition was well established for I_M inhibition by bradykinin (Gamper et al. 2005; Gamper and Shapiro 2003) or by P2Y receptor stimulation (Zaika et al. 2007), in which CaM was shown to be responsible. However, inhibition of CaM activation using dominant-negative CaM that cannot bind Ca²⁺ (Geiser et al. 1991) did not affect muscarinic inhibition of I_M (Gamper and Shapiro 2003; Zaika et al. 2007), leading to the conclusion that Ca²⁺ signaling is not involved in muscarinic inhibition of I_M. This conclusion was supported by the observation that in SCG neurons, oxo-M did not induce Ca²⁺ transients as robustly as bradykinin did (Cruzblanca et al. 1998; del Rio et al. 1999). In early experiments, however, muscarinic inhibition of I_M was shown to be suppressed when intracellular Ca²⁺ was heavily buffered (Beech et al. 1991). Further, Shapiro et al. (Shapiro et al. 2000) reported that oxo-M-induced Ca²⁺ transients are blocked by thapsigargin. Taken together, these findings implicate Ca²⁺ signaling in muscarinic receptor stimulation. Thus, we re-examined the roles of Ca²⁺ signaling in muscarinic inhibition of I_M inhibition.

First, we examined whether oxo-M could induce Ca²⁺ transients in SCG neurons and whether oxo-M-induced Ca²⁺ transients would be significantly smaller than bradykinin-induced Ca²⁺ transients, as previously described (Cruzblanca et al. 1998; del Rio et al. 1999). [Ca²⁺]_i was measured in SCG neurons using Fura 2-AM. As shown in Fig. 4, 10-μM oxo-M did induce Ca²⁺ transients in all SCG neurons tested though there was large cell-to-cell variation. On average, oxo-M-induced Ca²⁺ transients were not different from bradykinin-induced Ca²⁺ transients (Fig. 4). The peak amplitude of Ca²⁺ transient induced by oxo-M was similar to that induced by bradykinin. Paired t test confirmed that the effects of oxo-M on intracellular Ca²⁺ concentration were not significantly different from those of bradykinin in the same neurons (n = 3, p > 0.05). These results imply that the Ca²⁺-releasing powers of oxo-M and bradykinin are not significantly different.

Fig. 4

Muscarinic stimulation raises [Ca²⁺]_i in rat SCG neurons. SCG neurons were loaded with 2-μM Fura 2-AM for Ca²⁺ measurements. The F340/F380 ratio was used to estimate [Ca²⁺]_i. F340/F380 ratio (F´) changes in oxo-M or bradykinin-stimulated cells was summarized. Open circles indicate means ± S.E.M. Inset shows two representative [Ca²⁺]_i recordings of each group

Since muscarinic receptor stimulation elicited an increase of [Ca²⁺]_i, we tested whether Ca²⁺ mediates muscarinic modulation of I_M using 20-mM BAPTA. When the pipette solution contained 20-mM BAPTA, oxo-M minimally affected I_M (Fig. 5Aa). Superimposed deactivation tail currents showed that in a cell dialyzed with 20-mM BAPTA, I_M after 5 min oxo-M treatment was almost overlapped with that in controls. To rule out the possibility that I_M itself was reduced by 20-mM BAPTA, we obtained the steady-state current–voltage relationships for oxo-M-sensitive currents and XE-991-sensitive currents in BAPTA-loaded cells, using the same protocol as in Fig. 1. Figure 5Ac demonstrates the proportion of XE991-sensitive currents to total steady-state currents in 20-mM BAPTA-loaded cells was not reduced compared to that in 0.1-mM EGTA (52.40 ± 2.46% [n = 19] vs. control: 26.49 ± 1.41% [n = 7] at + 20 mV). The oxo-M-induced inhibition of I_M was determined as a proportion of oxo-M-sensitive currents to XE991-sensitive currents at + 20 mV as in Fig. 1 and was 22.21 ± 3.94% (n = 19, Fig. 5Ac), which was significantly smaller than that in controls (79.90 ± 13.62% (n = 7), p < 0.01). We then tested whether the effect of BAPTA on suppressing muscarinic inhibition of I_M could be attributed to inhibiting local Ca²⁺ signals. To do this, we tested the effect of EGTA on muscarinic inhibition of I_M. BAPTA is a fast Ca²⁺ buffer that effectively buffers all types of Ca²⁺ signals, whereas EGTA is a slow Ca²⁺ buffer that selectively spares local Ca²⁺ signals (Neher 1998). With 20-mM EGTA, muscarinic receptors were still able to inhibit M currents (Fig. 5Ba). The oxo-M-induced inhibition of I_M was calculated as a proportion of oxo-M-sensitive currents to XE991-sensitive currents at + 20 mV and was 103.05 ± 11.60% (n = 5, Fig. 5Bc), which was not significantly different from that of control conditions (p > 0.05). We confirmed that proportions of I_M to total outward currents in the presence of 20-mM EGTA (49.60 ± 3.48% [n = 4]) at + 20 mV) were similar to those in the presence of 20-mM BAPTA (52.40 ± 2.46% [n = 19]).

Fig. 5

Intracellular BAPTA, but not EGTA suppresses muscarinic modulation of I_M. The current inhibition induced by 10-μM oxo-M were measured in cells dialyzed with 20-mM BAPTA (A) or 20-mM EGTA (B). a, time course of tail current amplitude induced by the pulse protocol as shown in the inset. Right, superimposed deactivation tail currents before (black) or after application of oxo-M or XE991. b, families of current elicited by voltage steps from −60 mV to + 20 mV, in 10-mV intervals, before (left panel) and after application of 10-μM oxo-M (middle panel) or XE991 (right panel) in a cell shown in a. c, pooled data for normalized oxo-M-sensitive (closed circles) and XE991-sensitive steady currents (open circles) to control steady-state currents at + 20 mV, collected as in b

Next, we directly examined the effect of [Ca²⁺]_i on M currents in SCG neurons. We perfused cells with a bathing solution containing 10-μM ionomycin, a Ca²⁺ ionophore, and 10-mM CaCl₂ to raise [Ca²⁺]_i. We found that as [Ca²⁺]_i increased, the M current amplitude declined in parallel (SI 3). This is consistent with previous data showing the sensitivity of KCNQ2/KCNQ3 channels to [Ca²⁺]_i (Delmas and Brown 2005; Gamper and Shapiro 2003; Kosenko and Hoshi 2013). Taken together, these data suggest that Ca²⁺ signals play a critical role in M₁ muscarinic receptor-mediated regulation of M channels. The differential effects of EGTA and BAPTA imply that M channels are very close to Ca²⁺ sources and are regulated by local Ca²⁺ signals.

Discussion

We have demonstrated that muscarinic inhibition of I_M in rat SCG neurons is significantly suppressed by 20-mM BAPTA in pipette solutions (Fig. 5). In fact, similar findings were observed in earlier studies (Beech et al. 1991; Kirkwood et al. 1991). However, Ca²⁺ was not regarded as a candidate for couple muscarinic receptor activation to M channel inhibition, because oxo-M failed to increase the intracellular Ca²⁺ concentration in SCG neurons (Beech et al. 1991; del Rio et al. 1999). The current understanding of the distinction between muscarinic receptor-induced inhibition of I_M and bradykinin-induced inhibition of I_M relies, at least in part, on the inability of oxo-M to induce Ca²⁺ increase (Delmas and Brown 2005). However, some controversial reports stated that oxo-M induces Ca²⁺ increase in acutely dissociated rat SCG neurons (del Rio et al. 1999; Cruzblanca et al. 1998) and tsA-201 cells (Falkenburger et al. 2013). Furthermore, oxo-M was invariably shown to induce Ca²⁺ mobilization in other parts of the brain (Irving and Collingridge 1998; Seymour-Laurent and Barish 1995), and oxo-M-induced effects, such as GIRK channel inhibition, were shown to be mediated by Ca²⁺-dependent mechanisms (Sohn et al. 2007). Other reports exploring the PIP₂ depletion hypothesis showed that M current could be inhibited by muscarinic receptors in neurons overexpressing the neuronal calcium sensor, NCS-1 (Winks et al. 2005). NCS-1 up-regulates PI-4-kinase, which catalyzes the first step in PIP₂ synthesis from PI and requires Ca²⁺ to be activated (Koizumi et al. 2002; Rajebhosale et al. 2003; Zhao et al. 2001; Taverna et al. 2002; Burgoyne and Weiss 2001). In neurons overexpressing NCS-1, Winks et al. reported that M current inhibition by oxo-M was conserved, while the inhibitory effect of bradykinin was reduced (Winks et al. 2005). They claimed that oxo-M does not evoke enough Ca²⁺ release to activate NCS-1 for replenishing PIP₂ hydrolyzed by muscarinic stimulation. However, others reported similar basal Ca²⁺ levels, ~ 100 nM, in PC12 cells and SCG neurons, which are enough to activate NCS-1 in PC12 cells (Cruzblanca et al. 1998; Delmas et al. 2002; Meijer et al. 2014; Koizumi et al. 2002; Rajebhosale et al. 2003; Taverna et al. 2002). In this case, under basal conditions during muscarinic receptor activation, NCS-1 could compensate for PIP₂ consumption by PLC. If PIP₂ depletion hypothesis is true, in cells overexpressing NCS-1, oxo-M is unlikely to inhibit M current because NCS-1 replenishes PIP₂ but supporting our hypothesis that PIP₂ depletion is not the primary mechanism.

In the present study, we re-examined whether the inability of oxo-M to induce Ca²⁺ mobilization is a consistent finding. Surprisingly, we found that muscarinic receptor stimulation by oxo-M is capable of increasing intracellular Ca²⁺ concentration in rat sympathetic neurons. We noted that the cell-to-cell variation of the amplitude of Ca²⁺ transients was quite large; thus, in some cells, Ca²⁺ transients induced by oxo-M were small, which was similar to a previous report (Delmas and Brown 2002). However, this was also the case for B2R-induced Ca²⁺ transients (Fig. 4). Thus, we conclude that the current understanding of dual modulatory pathways for M channel regulation based on different Ca²⁺ mobilization powers, the ability to evoke changes of [Ca²⁺]_i, needs to be re-examined. We do not know the reason for the discrepancy of Ca²⁺ mobilization power of oxo-M among different studies, but differences in receptor density (Dickson et al. 2013; Falkenburger et al. 2013; Kruse and Whitten 2021), signaling microdomains that modify the efficacy of IP₃ to open its receptor (Zaika et al. 2011), or experimental conditions may be involved. Previous studies used voltage-clamped neurons with Fura 2 in pipette solutions and held the membrane potential near or below −60 mV during Ca²⁺ measurements (Beech et al. 1991; del Rio et al. 1999; Falkenburger et al. 2013; Dickson et al. 2013), whereas we used intact sympathetic neurons with Fura 2-AM. If Ca²⁺ entry through the voltage-dependent ion channels contributed to the oxo-M-induced [Ca²⁺]_i rises, this pathway might have been blocked by clamping the membrane potential hyperpolarized to −60 mV or below. The fact that removal of external Ca²⁺ prevented the rise of [Ca²⁺]_i by muscarinic receptor stimulation may support this possibility (Foucart et al. 1995).

In fact, it was also observed in earlier studies that high concentrations of Ca²⁺ chelator (20-mM BAPTA) can suppress muscarinic inhibition of I_M (Beech et al. 1991; Kirkwood et al. 1991). However, when 10-mM Ca²⁺ was added to 20-mM BAPTA-containing pipette solutions to increase free Ca²⁺ concentration from 12 to 143 nM, oxo-M was still capable of inhibiting I_M (Beech et al. 1991; Cruzblanca et al. 1998). These results were interpreted to mean that the effect of BAPTA to inhibit a muscarinic effect on I_M was attributable to lowering of resting [Ca²⁺]_i rather than inhibiting the rise of [Ca²⁺]_i by muscarinic stimulation (Beech et al. 1991). This interpretation was based on the assumption that adding 10-mM Ca²⁺ to 20-mM BAPTA-containing solution increases free Ca²⁺ concentration without affecting Ca²⁺ buffering power during the event of rising [Ca²⁺]. That is not the case, but Ca²⁺ buffering power is compromised because BAPTA is occupied by the added Ca²⁺, resulting in reduction of free BAPTA that is capable of buffering Ca²⁺ rise. Therefore, these results and our results may present evidence that Ca²⁺ signals that mediate muscarinic inhibition of I_M are only suppressed by a very high concentration of BAPTA. The requirement of high concentrations of BAPTA may imply two possibilities: muscarinic inhibition of I_M somehow requires minimum resting Ca²⁺, as was suggested previously (Beech et al. 1991), or muscarinic inhibition of I_M is mediated by local Ca²⁺ signals that can be suppressed by high concentrations of fast Ca²⁺ buffer, BAPTA. To distinguish these two possibilities, we tested 20-mM EGTA, which has similar equilibrium affinities for binding Ca²⁺ with BAPTA, but its Ca²⁺-binding rate is too slow to suppress dynamic Ca²⁺ rises. Since it is expected that resting Ca²⁺ concentrations should be lowered to the same extent by EGTA as it is by BAPTA, differences in the ability of BAPTA and EGTA to block Ca²⁺-triggered responses can be regarded to represent the involvement of local Ca²⁺ signals. Indeed, we found that muscarinic inhibition of I_M was potently blocked by BAPTA but completely insensitive to EGTA (Fig. 5), implying that the spatial proximity between the Ca²⁺ source of oxo-M-induced Ca²⁺ rise and Ca²⁺-sensitive target proteins that mediate I_M inhibition is so close that the coupling can only be blocked by very high concentrations of fast Ca²⁺ buffer. We calculated the length constant of Ca²⁺ microdomain in the presence of either BAPTA or EGTA, λ, which represents the mean distance that a Ca²⁺ ion diffuses before it is captured by a buffer molecule (Fig. 6). According to equation (7) by Neher (Neher 1998), λ = √(D_Ca/k_on [B]^o (k_on and [B]^o representing the rate constant of Ca²⁺ binding to the buffer and the concentration of free buffer, respectively). We referred diffusion coefficient of free Ca²⁺ ion (223 μm²/s) as reported by Allbritton et al. (Allbritton et al. 1992), and the values for k_on and the rate constant of Ca²⁺ binding to the buffer, to the study by Naraghi (Naraghi 1997), in which k_on for BAPTA and EGTA is 4.5 × 10⁸/M.s and 2.7 × 10⁶/M.s, respectively. λ is calculated to be about 5 nm and 60 nm in the presence of 20-mM BAPTA and 20-mM EGTA, respectively. Thus, our results imply that M channels are localized in the vicinity of the muscarinic receptor sensitive Ca²⁺ source within 60 nm, but away from this source at more than 5 nm. To prove this model, it is necessary to identify the Ca²⁺ source and Ca²⁺-sensitive target proteins involved in I_M inhibition. Further studies will be required to clarify this issue.

Fig. 6

Model for Ca²⁺ microdomains for muscarinic modulation of I_M in SCG neurons. The length constant of Ca²⁺ microdomains associated with individual M1 muscarinic receptors (M1) is calculated to be ~ 5 nm and 60 nm in the presence of 20-mM BAPTA (left) and 20-mM EGTA (right), respectively. Ca²⁺ rises in response to muscarinic stimulation can lead to weakening channel–PIP₂ interaction, resulting in the decrease of M currents

In addition, our results showed that muscarinic inhibition of M channel was not attenuated by blocking PIP₂ depletion with supply of PIP₂ (up to 200 μM), or by blocking PKC activation with a specific blocker, calphostin C, or by blocking both PIP₂ and PKC pathways. As other potential signaling pathways may have been involved, we examined whether G_βγ subunits of G protein or trafficking of channel proteins were involved in muscarinic inhibition of I_M. The results showed that neither seemed to have a critical role.

Our data demonstrate that PIP₂ depletion is not the ‘principal’ contributor to the inhibition of I_M by muscarinic signaling, in contrast to the PIP₂ depletion hypothesis for muscarinic action. Clearly, KCNQ channels are PIP₂ sensitive: they are directly activated by exogenous PIP₂ (Zhang et al. 2003). However, two distinct questions must be answered to evaluate the role of PIP₂ signals. The first is whether PIP₂ functions as a regulator of channel activity and a second, distinct question is whether PIP₂ signals play a role in M channel modulation by muscarinic receptors. The PIP₂ depletion hypothesis is based on a number of experiments focusing on the first question. For example, selective depletion of PIP₂ using an engineered chemical dimerization system almost completely suppressed the current, whereas PIP₂ directly applied to excised patches augmented the current (Suh et al. 2006; Zhang et al. 2003). These data, however, cannot rule out the possibility that during muscarinic signaling, PIP₂ depletion plays no role in decreasing current amplitudes. To determine the role of PIP₂ in receptor-mediated regulation of ion channels, specific experiments testing the role of PIP₂ in receptor-induced channel modulation are needed. To do this, we and others applied exogenous PIP₂ into neurons during G_qPCRs signaling. This experimental protocol was used to confirm the role of PIP₂ depletion in G_qPCR-mediated GIRK channel regulation (Cho et al. 2005, 2001; Meyer et al. 2001). As opposed to PIP₂-mediated GIRK channel regulation, muscarinic inhibition of KCNQ channels was not suppressed by PIP₂ (up to 200 μM, Fig. 2) and only partially reduced even by 500-μM PIP₂ (Robbins et al. 2006). These results underscore the notion that PIP₂ depletion might play only a small role in muscarinic inhibition of I_M. Another piece of evidence in favor of the PIP₂ model is that the dynamics and extent of channel inhibition by muscarinic receptors showed a close correlation to the translocation of the PH-GFP construct that provided a measure of overall PIP₂ hydrolysis (Hernandez et al. 2008b; Horowitz et al. 2005). However, the dynamics of PIP₂ hydrolysis does not necessarily correlate to the dynamics of PIP₂ depletion, especially in the proximity of a given ion channel. Since no method is available to measure changes in local concentrations of PIP₂, we developed a two-dimensional diffusion model to estimate them. Using this model, we found that the key to determining the spatial and temporal profiles of PIP₂ depletion is PIP₂ mobility (Cho et al. 2005), in that a profound PIP₂ depletion restricted to the microdomain adjacent to PLC occurs only when PIP₂ mobility is low. However, in general, diffusion constants for PIP₂ in neurons are not low (~ 0.5–2 μm²/s, (van Rheenen and Jalink 2002; Cho et al. 2005)) and there is no evidence that the physical properties of PIP₂ in SCG neurons are dissimilar from those of PIP₂ in other neurons. When PIP₂ is diffusible, the PIP₂ depletion induced by PLC activation is readily attenuated by diffusion, so that the resulting changes become slower and smaller (Cho et al. 2005; Cui et al. 2010). This is in contrast to the rapid and profound I_M inhibition observed during muscarinic stimulation. Support for the PIP₂ model also comes from the fact that the recovery from inhibition requires cytoplasmic ATP and is delayed by inhibition of the PI-4-kinase, which replenishes PIP₂ (Ford et al. 2003; Suh and Hille 2002). However, although recovery from inhibition requires resynthesis of PIP₂, whether the process of inhibition itself results directly from PIP₂ breakdown is unclear. It must be noted that for GIRK channels in HEK cells, the recovery from receptor-induced inhibition depends primarily on PIP₂ regeneration but receptor-induced inhibition results primarily from the change in the channel’s affinity for PIP₂ by other signaling molecules (Brown et al. 2005). Furthermore, as mentioned earlier, although muscarinic inhibition of I_M is reduced by overexpressing the synthetic enzyme PI-5-kinase (Winks et al. 2005), whether muscarinic receptor signaling no longer functions in cells with increased resting PIP₂ level caused by overexpression of PI-5-Kinase needs to be tested and resolved.

Reportedly, Ca²⁺ elevation does not require as much G_qPCR stimulation or local receptor density as does PIP₂ depletion or M channel inhibition (Falkenburger et al. 2013; Dickson et al. 2013; Kruse and Whitten 2021). Thus, Ca²⁺ signaling sensitizes SCG neurons to M₁R activation, allowing I_M inhibition even when M₁R is at low receptor density or under minimal muscarinic acetylcholine stimulation.

The results of our study do not exclude or support the possibility that PIP₂ depletion and Ca²⁺ elevation might occur simultaneously in response to muscarinic stimulation; and this could enhance the inhibitory effect. However, we do show that use of exogenous Ca²⁺ buffer to inhibit Ca²⁺ rises could block muscarinic inhibition, whereas exogenously restoring PIP₂ levels could not, suggesting Ca²⁺ elevation, not PIP₂ depletion, is the principal driver of muscarinic inhibition of M currents.