M3 muscarinic receptor activation of a delayed rectifier potassium current in canine atrial myocytes

Life Scicnns, Vol. 64, No. 21, pp. PL 2%257, 199!9 Copyight 0 1999 Elscvier Science inc. Printed in the USA. All rights sewed

PII SOO24-3205(99)00142-3

ELSEVIER

P?URiWCOLOGY LETTERS Accelerated Communication

Mj MUSCARINIC RECEPTOR ACTIVATION OF A DELAYED RECTIFIER POTASSIUM CURRENT IN CANINE ATRIAL MYOCYTES Hong Shi’, Huizhen Wang’, and Zhiguo Wang”2 ‘Research Center, Montreal Heart Institute, Montreal, Quebec HIT lC8, ‘Department of Medicine, University of Montreal, Montreal H3C 3J7, Quebec, Canada (Submitted November 10, 1998; accepted November 11, 1998; received in final form February 10, 1999) -

Abstract. Growing body of evidence indicates that the functional responses of cells to muscarinic acetylcholine receptors (mAChRs) are mediated by multiple receptor subtypes. It is commonly thought that the MZ receptor is the only functional mAChR subtype in the heart and little data regarding the potential roles of other subtypes in cardiac tissues has been reported. In the present study, we provide functional evidence for the presence and physiological function of an M3 receptor in canine atria1 myocytes. Using whole-cell patch-clamp techniques, we consistently found that pilocarpine, an mAChR agonist, induced a K+ current similar to but distinct from the classical delayed rectifier K” current. Same observations were obtained when choline or tetramethylammonium (TMA) was applied to the bath. The currents were abolished by 1 ILM atropine. Antagonists selective to Mi (pirenzepine, 100 nM), Ml (methoctramine 100 nM), or Mq (tropicamide 200 nM) receptors failed to alter the currents. Conversely, three different M3selective inhibitors, p-F-HHSiD (20-200 &I), 4-DAMP methiodide (2-10 nM) and 4-DAMP mustard (4-20 nM), all produced concentration-dependent suppression of the currents. A cDNA fragment representing the MX receptor was isolated from dog atria1 RNA and the mRNA level of this construct was 0.7 f 0.1 pg/pg total RNA, as quantified by the competitive RT-PCR methods. Our data strongly suggested that an Mj receptor exists and is coupled to a K’ channel in the heart. 0 1999 Elsevier Science Inc. Key Words p-F-HHSiD

mAChR, M, receptor, K+ current, cardiac cells, pilocarpine, choline, TMA, 4-DAMP,

Introduction Accumulating evidence indicates that the cellular responses to mAChRs are mediated by multiple receptor subtypes (1,2). mAChRs are subdivided based on the pharmacological profiles of receptor agonists and antagonists (1). To date, at least four different subtypes have been pharmacologically and functionally defined in primary tissues, designated Mi, Mz, M3, and M4. The Ml receptor is commonly believed to be the only functional mAChR subtype in the heart (3,4). Stimulation of cardiac M2 receptors by agonists like acetylcholine (ACh) is known to induce an inward rectifier K’ current (IKAc~) which contributes to parasympathetic regulation of heart rate and atria1 contractility, and of the action potential morphology and characteristics (5,6).

Corresponding author: Dr. Zhiguo Wang, Ph.D. Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, Quebec, Canada HlT lC8. Tel: (514) 376-3330, Fax: (514) 376-1355, E-mail: wanqz.@m.

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M3 receptors are found to be widely distributed in a variety of tissues, including brain, exocrine glands, various types of smooth muscles and vascular endothelial cells, etc (1,2). The functional role of M3 receptors has been well appreciated in many aspects, such as relief of apoptosis of cerebellar granule neurons, promotion of the secretion by exocrine glands, facilitation of smooth muscles contraction (gut, airways, blood vessels, etc.), and so forth. However, the presence and functions of M3 subtype in the heart have not been reported. In an effort to investigate muscarinic modulation of ion channels in canine atria1 myocytes, we obtained the functional evidence indicating that the stimulation of M3 receptors induces a delayed rectifier-like K+ current in the dog heart. Materials and Methods Patch-clamp techniques. Single canine atria1 myocytes were isolated with techniques as previously described (7). Patch-clamp techniques used in this study have been described in detail elsewhere (8). Ionic currents were recorded with an Axopatch-200B amplifier (Axon Instruments, CA). Borosilicate glass electrodes (1 mm O.D.) had tip resistances of l-3 MR when tilled with pipette solution. Junction potentials were zeroed before formation of the membrane-pipette seal in Tyrode’s solution. The capacitance was 79 f 3 pF (n=20) before and 75 f 2 pF after compensation, and the series resistance was 5.2 f 0.5 MS2 and 1.5 f 0.1 MQ before and after compensation, respectively. Experiments were conducted at 36+1”C. Solutions and drugs. The external solution for whole-cell patch-clamp recording had the following composition (mM): 136 NaCI, 5.4 KCl, 1 MgC12, 0.33 NaHzP04, 5 HEPES, 10 glucose and 1 CaC12; pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM): 0.1 GTP, 110 potassium aspartate, 20 KCI, 1 MgC12, 5 Mg-ATP, 10 HEPES, 10 EGTA, 5 phosphocreatine; pH adjusted to 7.3 with KOH. Contamination by sodium current was prevented by holding the cells at -50 mV. Potential contamination by other currents was minimized by including the following compounds in the bath solution: dofetilide (1 pM, to inhibit I xr, the rapid component of delayed rectifier K+ current), 293B (20 pM, to block I&, the slow component of delayed rectifier K+ current), glyburide (10 PM, to prevent ATP-sensitive K+ current), and Cd2+ (200 pM, to suppress Ca2+ current and Ca2+-activated transient outward Cl current). The current amplitude was measured as the magnitude of the time-dependent component and normalized to the cell capacitance to obtain the current density. To clone cDNA sequence of M3 Cloning of Mj Receptor cDNA Fragment from Dog Atrium receptor from dog heart, degenerate primers were designed based on published sequence of human m3. Reverse transcription (RT)-polymerase chain reaction (PCR) was used to obtain cDNA fragments from RNA samples extracted canine atria1 tissues. PCR products of desired size were gelpurified, subcloned and subjected to sequencing analysis. The procedures for synthesis of Competitive Reverse Transcriptase-Polymerase Chain Reaction RNA internal standards were same as previously described (9). Gene-specific primer (GSP, senseTGCAGGCCCAGAAGAGC and antisense-CCTTTTCCGCTTAGTGATCTG) pairs were designed based on the sequence of canine Ms cDNA fragment which we cloned. RNA mimic samples with serial 1O-fold dilutions were prepared and added to a constant quantity of sample RNA (total RNA of 1 pg for each reaction). RT was carried out in a 20-~1 reaction and the resulting firststrand cDNA (5 pl) was then used as a template for,PCR amplification in a 25-pl reaction mixture with High Fidelity PCR kit (Boehringer Mannheim). Reactions were hot-started at 94”C, and continued for 3 min of initial melting. The cycling profiles were 30 set denaturing at 94’C, 30 set

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annealing at 53°C and 40 set extension at 72’C, for 30 cycles, followed by a final extension step of 5 min at 72% Densitometry was employed for quantification of PCR products. Equal input of the initial amount of sample RNA, and equal efficiency of PCR amplification for sample RNA and the internal standard RNA were verified with methods described previously (9). Data analysis. Group data are expressed two groups were performed on raw data PcO.05 taken to indicate a statistically were performed with analysis of variance

as me&standard error. Statistical comparisons between with paired or unpaired Student’s t-test, with a two-tailed significant difference. Comparisons for multiple groups with Scheffe’s contrasts. Results

While studying modulation of cardiac ion currents by pilocarpine, an mAChR agonist (IO), we unexpectedly but consistently observed an outward current elicited with depolarizing voltage steps only in the presence of this agent, which was otherwise absent in canine atria1 myocytes. The outward current was current disappeared upon washout of pilocarpine. Pilocarpine-induced characteristic of delayed rectifier K’ current with time-dependent activation to a maximum level during depolarization (-40-+50 mV) and a deactivation tail current upon repolarization to -30 mV. The I-V curve was outwardly rectifying at test potentials between -40 and +50 mV (the middle panel of Fig. 1). The size of the currents depended on the concentration of pilocarpine, as shown in the right panel of Fig. 1. The ECX, value, obtained by fitting the fractional currents (currents measured at varying concentrations relative to the current amplitude with 10 pM pilocarpine) at varying concentrations to Hill equation, was 3.1 pM with a Hill co-efficient of 4. Our data also confirmed that the currents were mainly carried by potassium ions (data not shown).

The currents were observed either in the absence or in the presence of channel blockers (glyburide, dofetilide, 293B and Cd’+) and were not affected by any of these compounds, suggesting that pilocarpine-induced currents were not related to 1~ and 1~ or any other ion currents, but probably represent a novel current similar to the recently described K’ current induced by choline in dog atrium (11). We found that choline (10 mM, a concentration causing maximal level of current induction) activated a current with characteristics identical to pilocarpine-induced current (see Fig. 1B). Similarly, TMA (tetramethylammonium, 0.5 mM, a concentration causing maximal inductionof the current), which has been shown to be an mAChR agonist (12) with selectivity to M3 subtype (13) was also able to induce the same current (Fig. 1C). Atropine (1 PM), a nonselective mAChR antagonist, quickly abolished the currents induced by pilocarpine, choline, or TMA, respectively. Similar results were observed in total of 5 cells for pilocarpine, 8 cells for choline and 7 cells for TMA. The atropine data indicate that stimulation of mAChRs is required for the activation of the current. We therefore further explored the mAChR subtype specificity of K’ channel coupling, by using various pharmacological probes selective to different mAChR subtypes. Effects of several mAChR antagonists on the current were evaluated, including pirenzepine (100 nM, for Ml subtype) (2,14), methoctramine (20-100 r&l, a cardiac-selective Mz inhibitor) (2, 15), tropicamide (200 nM, for M4) (2, 16) and p-F-HHSiD (hexahydro-sila-hydrochloride, p-fluoro analog, 20-200 nM, an Mlselective antagonist) (17) plus 4-DAMP methiodide (4-Diphenylacetoxy-N-methylpiperidine, 2-10 r&I, a competitive MJ inhibitor) (2,18) and 4-DAMP mustard (4-20 nM, an irreversible M3selective inhibitor) (19). The drug concentrations representing optimal subtype selectivity were chosen based on previous studies. None of the inhibitors for Ml, Mz and Mb subtypes produced appreciable effects on the currents (P>O.O5) induced by any one of the three agonists (Fig. 2B). In contrast, the current amplitude was substantially diminished by all three M3-selective antagonists, while the kinetics was left unaltered. The currents blocked by 4-DAMP methiodide (Fig. 1) and p-

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Fig. 1 Activation of K’ currents via simulation of mAChRs in canine atria1 myocytes. Currents were elicited by voltage protocols shown in the inset of panel A. (A) Currents activated by pilocarpine (Pilo, 10 PM). Left panel: raw traces elicited at +50 mV; middle panel: I-V relationships of pilocarpine-induced currents before and after 4-DAMP. *P
F-HHSiD (Fig. 2A) were completely converted back to control values upon washout of these chemicals. The effects of 4-DAMP mustard, however, were irreversible. These results suggested that the K” channel was coupled to the M3 receptor and the current was activated by Mx receptor stimulation by pilocarpine, choline or TMA. For comparison, the ACh-activated, Mz-mediated inward rectifier K+ current (Ik& was also recorded (Fig. ID). ACh (1 pM) induced an outward current which decayed rapidly with time at +50 mV (Fig. 1D) and inward currents at potentials negative to -80 mV (data not shown). Addition of methoctramine (100 r&I) suppressed the inward currents elicited with hyperpolarizing pulses but increased the outward current at depolarized potentials. And this outward current was markedly reduced by concomitant application of 4DAMP (2 nM, Fig. 1D). The inset of Fig. 1D shows the ACh-induced, 4-DAMP-sensitive current. Similar results were observed in 4/7 cells. Methoctramine alone failed to induce any outward currents in the absence of ACh.

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C

A Pilocarpine

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01234567

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pF.HHSiD 4DWP 4-DAMP 20 200 methialide rmstard 2 10

4 20

xa’

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Fig. 2 mAChR agonist-induced K’ currents are mediated by M3 receptors in canine atria1 myocytes. (A) Raw current traces obtained from a representative experiment, showing the effects of p-F-HHSiD (20 nM) on pilocarpine (10 pM)-induced currents and recovery of the depressed currents after washout of p-F-HHSiD.(B) Effects of M3antagonists and antagonists selective to other subtypes on pilocarpine-induced currents elicited at +50 mV, expressed as percent reduction of the currents with drugs over control values. PZ-pirenzepine for MI; Meth- methoctramine for Mz; and Troptropicamide for M,+ Drug concentrations (in nM) are provided with values following each drug name. The number of cells used for analyses is three for all cases. *P
To ensure that the observed effects of M3 antagonists on pilocarpine-induced currents were devoid of non-specific actions on subtypes other than M3 receptors, we performed additional experiments by protecting Mr/Mzm receptors by pre-incubating the cells with the antagonist cocktail containing pirenzepine, methoctramine and tropicamide for 10 minutes before adding agonist. Then pilocarpine was applied to antagonist cocktail-containing solution to activate the currents.

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The current density so measured was not significantly different from that measured in the absence of the antagonist cocktail (data not shown). Subsequently, M3-selective antagonists were added to the bath. Our data clearly demonstrated that the ability of M3 antagonists to suppress the currents was preserved while the MliAWM4 receptors were inhibited. To obtain further evidence at the molecular level in support of our functional studies for the presence of Mj receptors in the heart, we cloned a 432 bp cDNA fragment representing M3 mAChR from dog atrium (accession No. AFO56305). This fragment, spanning part of the third intracellular loop between transmembrane domains 5 and 6 thought to contain critical determinants of G protein coupling specificity, shares 8 1.5% homology to the same region of human brain MJ at the amino acid level. Primers were designed based on the sequence of the cloned fragment and used for detection and quantification of the mRNA encoding an M3 receptor. The quantity of m3 transcript analyzed by competitive RT-PCR from five atria1 samples was 0.7 f 0.4 pg mRNA@g total RNA, indicating an abundant expression. mRNA level of m2 was found approximately 5-fold higher than that of m3 in our samples. An example of competitive RT-PCR analysis for m3 mRNA is shown in Fig. 2C. The absence of contamination of our RNA samples by sources from neurons, vascular smooth muscles and fibroblasts was confirmed by methods described previously (9).

Discussion Our study provided for the first time the functional evidence for the presence receptor subtype in the heart. This implies that the structures and functions heart might actually be more diverse than what is commonly thought. This opens up new opportunities for better understanding the parasympathetic function and the interactions between the receptor and channel proteins.

and function of M3 of mAChRs in the finding potentially control of cardiac

Our conclusion relied largely on pharmacological dissection, thus imperfect specificity of mAChR antagonists could have obscured the accurate analysis of the data. Nonetheless, similar approach has been widely and successfully used for identifying and classifying mAChR subtypes. Pirenzepine is a well-recognized Ml-selective antagonist (2,14). Methoctramine is known to have an affinity two orders of magnitude higher at an M2 receptor than at other subtypes, particularly in cardiac tissues (2,15). Similarly, tropicamide has a higher affinity to M4 subtype than to others (16). The fact that these above-mentioned compounds failed to alter the currents made it unlikely that M,lM2/1L14receptor subtypes contributed significantly to the currents induced by pilocarpine, choline, or TMA. On the other hand, both 4-DAMP (2,18,19) and p-F-HHSiD (17) are selective for M3 receptors when used at appropriate concentrations. Inhibition of the currents by any one of these antagonists revealed the existence and physiological tinction of M3 subtype in cardiac cells. It is known that stimulation of cardiac M2 receptors induces an inward rectifier K+ current which is biophysically and pharmacologically distinct from the currents induced by pilocarpine, choline or TMA. As shown in Fig. 1, the M3-mediated currents possess delayed rectifier properties and outward rectifying I-V relationships, whereas the Mz-mediated current (IKAu,) demonstrated timedependent relaxation (Fig. 1D) and strong inward rectification (I-V not shown). This further suggests that the MZ receptors are unlikely contribute to the currents activated by these agonists. A study performed by Fermini and Nattel demonstrated that choline induced a K+ current via stimulation of an unknown subtype of mAChRs and our study clarified the subtype specificity of mAChR (MJ) coupling to the K+ channel. TMA has been reported to slow the sinus rate and to weaken the contraction of rat hearts (20). Our results revealed a possible mechanism underlying, at least in part, the TMA-produced negative inotropic and chronotropic effects. The sensitivity to 4DAMP of the outward currents induced by ACh in the presence of methoctramine suggests that ACh can also activate the M3-mediated Kf currents. However, we do not know why this component was seen only when M2 receptors had been largely suppressed by methoctramine. Further studies are absolutely required to clarity this issue.

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The fact that the cDNA fragment representing m3 isofonn was isolated from canine atrium and that the abundant expression of mRNAs coding for M3 subtype was quantified by competitive RTPCR methods adds an additional evidence for the presence of M3 receptor in the canine atrium. It is speculated based on our data that an M3 receptor might play a role in determining membrane repolarization of atria1 cells owing to its ability to induce a K+ current. Whether M3 receptors also regulate other ion channels warrants further studies. The potential roles of M3 receptors in regulating the cardiac functions and the pathogenesis of heart disease await to be established. Acknowledgment This work was supported in part by the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, an Establishment Grant for young investigators from the Fonds de Recherche en Sante de Quebec awarded to Dr. Wang, and the Fonds de la Recherche de 1’Institut de Cardiologie de Montreal. Dr. Wang is a research scholar of the Heart and Stroke Foundation of Canada. The authors wish to thank XiaoFan Yang for excellent technical assistance. References

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