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Tetraethylammonium potentiates the activity of muscarinic potassium channels in guinea-pig atrial myocytes

Desuo Wang and David L. Armstrong

	ABSTRACT

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1. The modulation of native muscarinic potassium channels (K_ACh) by tetraethylammonium (TEA) was studied at 35^oC in cell-free patches from acutely dissociated guinea-pig atrial myocytes. The channels were identified unambiguously by their conductance, inward rectification, rapid gating kinetics and pharmacological responses to muscarinic agonists and GTPS.

2. Addition of 5 mM TEA to the cytoplasmic side of the patches almost doubled the open probability of K_ACh channels that had been activated maximally by GTPS. In contrast even 30 mM TEA did not significantly potentiate the response to carbachol in whole-cell recordings.

3. Unlike GTPS, TEA alone did not activate K_ACh channels de novo, but in patches that showed spontaneous K_ACh activity, 5 mM TEA increased channel open probability fourfold in the absence of added sodium, ATP or guanine nucleotides. Furthermore, the effect of TEA was not blocked by 10 M atropine or by 1 mM GDPS, and subsequent addition of 0.1 mM GTPS did not stimulate channel activity further in the presence of TEA.

4. Phosphatidylinositol 4,5-bisphosphate (PIP₂) also stimulates K_ACh channels under these conditions, but the kinetics of gating differ from channels stimulated by either TEA or GTP, which are very similar to one another.

5. The effects of TEA were not mimicked by tetramethyl- or tetrapentylammonium or by sodium or spermine, and TEA did not potentiate the activity of other inwardly rectifying potassium (K_ATP) channels in patches from cardiac myocytes.

6. We consider the possibility that TEA is mimicking the effect of an unidentified cellular factor, not sodium or PIP₂, which normally occupies the TEA site on K_ACh channel proteins but which diffuses away when the patch is excised.

	INTRODUCTION

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K_ACh channels are a specific heteromeric class (Kir 3.1 and 3.4) of inwardly rectifying potassium channels (Sakmann et al. 1983; Krapivinsky et al. 1995), which regulate the excitability of atrial and nodal myocytes in the heart in response to muscarinic receptor stimulation (Breitwieser & Szabo, 1985; Pfaffinger et al. 1985). In addition to the well-known stimulation by G proteins (reviewed by Yamada et al. 1998), both native and recombinant Kir 3.1/3.4 channels are also stimulated in cell-free patches by sodium ions (Sui et al. 1996; Ho & Murrell-Lagnado, 1999) and by phosphatidylinositol phosphates (Huang et al. 1998; Sui et al. 1998), but the interdependence of these effects and their role in muscarinic stimulation of native K_ACh channels have not been established conclusively (Kim & Bang, 1999; Petit-Jacques et al. 1999). Nevertheless, drugs that open potassium channels have been useful in treating human disease (Belardinelli et al. 1995; Lawson, 1996). Here we report a novel mechanism for selectively stimulating K_ACh channels with tetraethylammonium (TEA) that is distinct from the previously described effects of sodium and PIP₂ on K_ACh activity. This effect is particularly surprising because TEA is a well-known blocker of many potassium channel pores (Armstrong, 1974; Stanfield, 1983), but novel, membrane-permeable drugs that target only the intracellular stimulatory site of TEA action might prove useful in treating some forms of heart disease.

	METHODS

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Atrial myocyte isolation

Atrial myocytes were dissociated by a method described previously (Wang & Belardinelli, 1994) using collagenase Type I (Worthington Biochemical) from the hearts of guinea-pigs (Hartlet strain, weighing 250-350 g), which had been killed after deep anaesthesia by inhalation of methoxyflurane according to the guidelines of the National Institute of Environmental Health Sciences Animal Care and Use Committee (study 97-02). Dissociated atrial cells were collected and stored at 20°C in a modified Tyrode solution containing 0.1 mM calcium until transfer to the experimental chamber. The standard Tyrode solution contained (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.53 MgCl₂, 0.33 NaH₂PO₄, 5.5 glucose and 5.5 Hepes (pH 7.4 with NaOH). Inside the recording chamber the myocytes were superfused with solution at a rate of 2-4 ml min^-1. All recordings were made at 35°C unless specifically indicated. Only quiescent, rod-shaped myocytes with clear striations were chosen for experiments.

Electrophysiology

Patch pipettes were pulled from borosilicate glass capillaries (Corning no. 7520) and connected to the headstage of an Axopatch 200B amplifier (Axon Instruments, Inc.). Whole-cell currents were recorded from cells bathed in a solution that contained (mM): 130 NaCl, 15 KCl, 2 MgCl₂, 0.5 CaCl₂, 0.33 NaH₂PO₄, 1.0 BAPTA, 5.5 glucose and 5.5 Hepes. The cells were voltage clamped through conventional ruptured patches with pipettes that contained (mM): 140 KCl, 2.0 MgCl₂, 0.5 CaCl₂, 1.0 BAPTA and 5.0 Hepes. In some experiments (Fig. 1B), 30 mM KCl of pipette solution was replaced with 30 mM TEA. The membrane potential was held at -40 mV, and whole-cell current was elicited every 3 s with a 1 s voltage ramp from -120 to +40 mV. Single channel activity was recorded using pipettes filled with a solution containing (mM): 140 KCl, 1.0 CaCl₂, 2.0 MgCl₂ and 5.0 Hepes (pH 7.4 with KOH). In several experiments 10 M atropine and 100 M glibenclamide were also added to the pipette solution. Cell-attached and cell-free (inside-out) patches with seal resistances > 5 G were formed in a bath solution containing (mM): 140 KCl, 2.0 MgCl₂, 5.0 Hepes, 0.5 CaCl₂ and 1.0 BAPTA. Patches were held at 0 mV for stability and stepped to the indicated voltage for 0.5 s at 6 s intervals (Hamill et al. 1981). Carbachol and guanine nucleotides were dissolved in water and stored as concentrated solutions at -20°C. One molar tetraethylammonium chloride (Baker and Sigma Chemical companies) was solubilized in water. Proton NMR spectra of samples from this stock solution were obtained on a Varian UNITY 500 spectrometer operating at 500.18 MHz (E. F. DeRose, R. London & D. Wang, unpublished data).

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Figure 1 Tetraethylammonium (TEA) potentiates KACh channel activity stimulated by GTPS A and B, whole-cell currents from atrial myocytes at 35°C elicited by 1 s voltage ramps from -120 to +40 mV. Bath application of 10 M carbachol (2) increases control (1) currents in the absence (A) or presence (B) of 30 mM TEA in the pipette solution. C, the effects of 5 mM TEA and 20 M GTPS on channel activity at 20°C from an inside-out patch at -90 mV plotted as NPo of inward currents. Note that TEA does not increase KACh activity until the channels have been activated by GTPS. D, representative traces from the experiment in C.

Data collection and analysis

Records were filtered at 2 kHz, digitized at 10 kHz and acquired and analysed using pCLAMP 6.03 software (Axon Instruments). The histogram of channel open durations was fitted to a two-exponential function (Simplex-LSQ; Axon pSTAT 6). Average channel activity (NP_o), which represents the average open probability (P_o) of an individual channel multiplied by the number (N) of active channels in the patch, was measured over a 300 ms duration in each voltage step and plotted versus time. NP_o was measured as the mean value over at least 30 consecutive steps (i.e. 3 min) during the treatment indicated. Error bars show the standard error of the mean from n patches. Significance was determined using Student's paired t test or one-way ANOVA.

	RESULTS

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TEA potentiates K_ACh channel stimulation by GTPS but not by carbachol

Figure 1A shows a typical response of whole-cell currents to muscarinic receptor stimulation in physiological saline. Adding 30 mM TEA to the patch pipette (Fig. 1B) significantly reduced membrane conductance at all voltages and completely blocked carbachol-stimulated outward current. Nevertheless, the absolute increase in inward current produced by subsequent application of carbachol was not altered significantly by TEA, which increased the mean from 717 ± 270 to 785 ± 205 pA (n = 3). Therefore, we were surprised to observe that adding TEA to the cytoplasmic side of guinea-pig atrial myocytes dramatically potentiated the response of K_ACh channels to GTPS (Fig. 1C and D).

TEA alone could not activate K_ACh channel activity in cell-free patches when basal open probability (NP_o) was less than 0.01 (n = 0/5), but TEA almost doubled the K_ACh activity elicited by GTPS (n = 21/21). Even maximally effective concentrations of GTPS (ēgeģ 20 M) produced 80% greater increases in open probability when 5 mM TEA was present. Inhibition of muscarinic receptors with 10 M atropine in the pipette solution did not prevent the potentiation by TEA from the cytoplasmic side of the membrane (not shown). Thus, TEA did not produce its effects by stimulating muscarinic receptors. Although atrial myocytes express at least three distinct classes of inwardly rectifying K⁺ channels, K₁, K_ACh and K_ATP (Barry & Nerbonne, 1996), the channels stimulated by TEA were identified unambiguously by their conductance (~40 pS), inward rectification, rapid gating kinetics (_open < 1 ms) and pharmacological responses to muscarinic agonists and GTPS (Yamada et al. 1998).

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Figure 2 Tetraethylammonium (TEA) rapidly and reversibly potentiates spontaneous KACh channel activity without adding ATP or guanine nucleotides A, representative traces showing effects of 5 mM TEA on unitary KACh currents at -100 and +100 mV. Patches were voltage clamped in symmetrical 140 mM KCl solutions at 35°C with BAPTA and no Mg2+ on the cytoplasmic side of the membrane. B, channel activity from another patch at -90 mV plotted as NPo of inward currents. C, channel activity at +90 mV plotted as NPo of outward currents.

TEA potentiates spontaneous K_ACh activity

In approximately one-third of the patches that were excised into nucleotide-free salt solutions at 35°C the average open probablility (NP_o) of K_ACh channels increased spontaneously from below 0.001 to 0.27 ± 0.07 (n = 19). The increase in activity occurred within 5 min, even in the presence of 1 mM GDPS to inhibit spontaneous G protein activation (n = 3/3), and remained stable for the lifetime of the patch, up to 30 min. Both inward and outward K_ACh channel activity could be recorded from these patches by omitting Mg²⁺ from the bath solution to reduce rectification (Vandenberg, 1987; Matsuda et al. 1987). Addition of 5 mM TEA to the cytoplasmic side of such patches blocked outward current through the channels but rapidly and reversibly increased NP_o from 0.27 ± 0.07 to 1.12 ± 0.25 at negative voltages (n = 16/19). The potentiation by TEA was observed in the absence of any added sodium, ATP, PIP₂ or guanine nucleotides (Fig. 2). Thus, the potentiation by TEA is distinct from all previously reported effects on K_ACh channel activity. Nevertheless, the additional channel activity produced by TEA was indistinguishable from the channel activity elicited in patches from the same cells by carbachol and GTP (Fig. 3), and GTPS could not augment the activity further after maximal stimulation by TEA (not shown).

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Figure 3 TEA potentiates channel activity with the same kinetics and conductance as the channels stimulated by GTP A, representative single channel records during steps from 0 mV to the voltage indicated in 3 different inside-out patches at 35°C exposed to 5 mM TEA, 200 M GTP, or 50 M PIP2. B, open dwell-time histograms obtained from the channel openings to the first level. Results of the fit to a two-exponential function (Simplex-LSQ; Axon pSTAT 6) are given. PIP2, but not TEA or GTP, markedly increased the open time duration in a fraction of the population. C, current-voltage relationship for the channels in A. Note that the channels in all three patches have the same inwardly rectifying conductance, ~40 pS.

Other amines do not mimic TEA action

To investigate the specificity of TEA action we applied several other amines to the bath solution during inside-out recordings of cell-free patches with spontaneous K_ACh activity. Neither ammonium chloride (up to 10 mM) nor spermine (up to 0.5 mM) stimulated K_ACh channels, but subsequent addition of TEA to the same patches stimulated activity at negative voltages with half-maximal stimulation at 2.5 mM TEA (Fig. 4). In contrast, tetramethyl- and tetrapentylammonium (TMA and TPA) inhibited K_ACh activity at all concentrations we examined (Fig. 4). Sodium ions have been reported to stimulate K_ACh channels (Sui et al. 1996; Ho & Murrell-Lagnado, 1999), but in the absence of ATP even unphysiologically high concentrations (> 20 mM) of Na⁺ only occasionally stimulated K_ACh activity under our conditions (n = 2/6). Furthermore, the increases produced by sodium were much smaller than those produced by TEA in the same patches (Fig. 4). Other K⁺ channel inhibitors, such as Cs⁺, Ba²⁺ and 4-aminopyridine, produced no stimulation of K_ACh activity (not shown).

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Figure 4 Specificity of TEA action on KACh channels Dose-response data for KACh channel modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), sodium ions (Na), spermine, and tetraethyl- (TEA), tetramethyl- (TMA) and tetrapentyl- (TPA) ammonium. The stimulatory effects of TEA, Na+ and PIP2 were normalized to the maximal stimulation by 15 mM TEA. The inhibitory effects of TPA and TMA were normalized to the basal channel activity. Each data point represents 3-12 experiments; error bars indicate standard error of the mean when it is larger than the symbol.

TEA and PIP₂ stimulate K_ACh through different mechanisms

The only molecule we tested that was as effective as TEA at stimulating spontaneous K_ACh activity in the absence of guanine nucleotides was the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂). As reported previously by others (Huang et al. 1998; Sui et al. 1998), PIP₂ directly stimulated K_ACh activity (Fig.4). However, we noticed that PIP₂ and TEA produced qualitatively distinct effects on the kinetics of native K_ACh channels (Fig. 3). The kinetics of TEA-stimulated channels are very similar to the kinetics of channels stimulated by GTP in the presence of carbachol (Fig. 3). In contrast, PIP₂ also increases the mean duration of channel opening (Fig. 3). Thus, it is unlikely that PIP₂ mediates the stimulation of K_ACh channels by TEA.

TEA does not stimulate other inwardly rectifying channels

K_ATP channels are a closely related family of inwardly rectifying potassium channels (K_IR 6.2/SUR2A), which are known to be activated by small molecules such as cromakalim and pinacidil (Inagaki et al. 1996; Babenko et al. 1998) and by PIP₂ (Baukrowitz et al. 1998; Shyng & Nichols, 1998) but not directly by GTPS or G protein subunits (Ito et al. 1992). Therefore, we tested the effect of TEA on K_ATP channels in inside-out patches from ventricular myocytes, which do not express K_ACh channel proteins at detectable levels (Kubo et al. 1993). Bath application of 5 mM TEA blocked inward currents through K_ATP channels (Fig. 5), which were identified unambiguously by their large unitary conductance (~78 pS), bursting open-close kinetics, and activation by 1 mM uridine diphosphate (Tung & Kurachi, 1991). Therefore, the effect of TEA is selective for the K_ACh channels.

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Figure 5 TEA blocks inward current through KATP channels in inside-out patches from ventricular myocytes A, bath application of TEA inhibits both basal and UDP-stimulated KATP channel activity, plotted as NPo versus time. B, at each of the time points indicated, 10 consecutive traces of channel activity during 500 ms steps to -90 mV are superimposed.

Evidence for a cellular factor acting like TEA on K_ACh channels

As others have shown previously (Kurachi et al. 1986; Ito et al. 1991), exogenous guanine nucleotides do not restore K_ACh activity in cell-free patches to the same level that muscarinic agonists achieve in cell-attached patches (Fig. 6). For example, in the presence of 5 M carbachol in the pipette solution, a maximally effective concentration (200 M) of GTP only increased K_ACh activity on average to 58% of carbachol's effect before patch excision (Fig. 6D). Interestingly, subsequent addition of TEA fully restored K_ACh activity (Fig. 6A, B and D). Similarly, maximally effective concentrations of GTPS (20 M) did not fully activate K_ACh channels in the absence of agonists, but TEA further increased the channel activity in the same patches exposed to GTPS (Fig. 6C and D). Nevertheless, maximal stimulation by GTPS and TEA together never exceeded the level of activity observed in cell-attached patches stimulated with muscarinic agonists, which is consistent with the lack of effect of TEA on carbachol-induced currents recorded in the whole-cell configuration (Fig. 1A).

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Figure 6 TEA potentiates KACh channel activity in the presence of maximally effective concentrations of guanine nucleotides A, representative current traces at both -90 and +90 mV from a single patch exposed to the indicated reagents. B and C, time course of NPo measured at -90 mV with (B) or without (C) 5 M carbachol (CCh) added to the pipette solution. D, average data from 4 patches stimulated with CCh and GTP and 16 patches stimulated by GTPS. Note that addition of TEA increases channel activity to the same level as that recorded under cell-attached patch configuration when the pipette contained CCh.

	DISCUSSION

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Here we document a novel and selective potentiation of K_ACh channel activity by TEA in cell-free patches. The channels were identified unambiguously by their conductance, inward rectification, rapid gating kinetics and pharmacological responses to muscarinic agonists and GTPS. Although TEA alone did not activate silent channels, nor did it potentiate the response to muscarinic agonists, it was more effective than guanine nucleotides at potentiating activity in cell-free patches once the channels had been activated. These results are particularly surprising because TEA is a well-known pore blocker of many K⁺ channels (Armstrong, 1974; Stanfield, 1983). TEA also blocked current flow through the K_ACh channels in our experiments (Fig. 2), but with TEA on the cytoplasmic side of the patch, only outward currents were blocked, and the potentiation was observed as an increase in the frequency of unitary inward currents at voltages more negative than the potassium equilibrium potential (E_K). We attribute this effect to TEA and not to any unidentified contaminants of the commercially available compound because we could not detect any significant level (> 10 nM) of organic contamination of the TEA stock solutions by NMR spectroscopy (see Methods). Furthermore, closely related quaternary amines (TMA and TPA) had only inhibitory effects on K_ACh channels (Fig. 4). Finally, TEA did not stimulate other inwardly rectifying potassium channels in guinea-pig cardiac myocytes (Fig. 5). Thus, the potentiation of K_ACh channel activity by TEA is highly selective, both for the ligand and for the target.

Although we have not directly demonstrated TEA binding to the channel protein, the rapid, robust and reversible response to TEA in cell-free patches in the absence of sodium or of any exogenous nucleotides (Fig. 2) indicates that the TEA binding site must be closely associated with the channel protein in the membrane. In addition neither atropine nor GDPS blocked the potentiating effect of TEA, which also rules out TEA producing its effects through muscarinic receptor or G protein stimulation. In fact TEA was more effective than maximal concentrations of hydrolysis-resistant GTP analogues at potentiating K_ACh channel activity. However, unlike GTPS, which activates K_ACh channels in 95% of all cell-free patches, TEA alone never activated channels de novo. Therefore, we are left with the hypothesis that the stimulatory site for TEA is only accessible when the channel is gating.

Nevertheless, the potentiation of K_ACh activity by TEA does not appear to require either G proteins or phosphatidylinositol phosphates (PIP₂). In the absence of exogenous PIP₂, activation of K_ACh by G subunits requires Mg-ATP (Sui et al. 1998); however, we observed potentiation of spontaneous K_ACh activity by TEA without adding any ATP or guanine nucleotides (Fig. 2). Spontaneous run-up of K_ACh activity in nucleotide-free solutions has also been reported for excised patches from frog atrial myocytes at 20°C (Otero et al. 1998). In those experiments, the increase in activity was much smaller (NP_o < 0.05) than we observed in excised patches from guinea-pig myocytes at 35°C and was attributed to spontaneous release of subunits. This is unlikely to be the explanation for the spontaneous activity we observed because subsequent addition of GTPS further stimulated channel activity (Fig. 6C) and 1 mM GDPS did not prevent the run-up. Other investigators have reported G protein-independent stimulation of K_ACh channels by sodium ions (Sui et al. 1996), but this effect required the presence of Mg-ATP and unphysiologically high concentrations of sodium ions (EC₅₀ ~40 mM) and was associated with an increase in open channel lifetime from ~1 to ~4 ms. In contrast the effect of TEA was observed in solutions to which no ATP or sodium had been added (Fig. 2), and was not associated with any increase in open channel lifetime (Fig. 3).

Even maximally effective concentrations of guanine nucleotides did not fully restore K_ACh activity in cell-free patches to the same level that muscarinic agonists achieved in cell-attached patches unless TEA was added to the cytoplasmic side of the membrane (Fig. 6). On the other hand TEA produced exactly the same level of activity as muscarinic agonists and was not potentiated further by subsequent addition of guanine nucleotides. Therefore, either muscarinic receptors normally stimulate K_ACh channel activity through two pathways, only one of which can be activated by guanine nucleotides in cell-free patches, or an endogenous cellular factor of intact atrial myocytes normally occupies the site at which TEA produces its stimulatory effects. In either case, any membrane-permeable drug that stimulated K_ACh channels without blocking the pores of other potassium channels would be an interesting candidate to test in the treatment of human cardiac disease.

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Acknowledgements

We are grateful to Xinghua Lu at the University of Pittsburgh for writing a software routine to automate NP_o plots in Origin from data in pCLAMP, and to Eugene F. DeRose and Robert London at NIEHS for testing the purity of the quaternary ammonium solutions by NMR spectroscopy.

Corresponding author

D. L. Armstrong: LST (F2-05), NIEHS, 111 Alexander Drive, RTP, NC 27709, USA.
Email: armstro3{at}niehs.nih.gov

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