Introduction

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In the last few years, the attention of several laboratories has been focused on the investigation of the cellular mechanisms by which taurine, a well known sulfonic amino acid abundantly distributed in different tissues including skeletal muscle, exerts its effects. This comes from the fact that taurine shows several interesting properties, such as antioxidant, osmo-regulator control of the Ca²⁺ homeostasis and antischemic properties (Huxtable and Sebring, 1986; Conte Camerino et al., 1989; Pasantes-Morales et al., 1998). "In vivo" studies have also shown that taurine antagonizes the age-dependent impairment of the contractility function of skeletal muscle fibers in aged rats (Pierno et al., 1998).

Emerging evidence indicates that taurine exerts its actions by interacting with different ion channels, including K⁺ channels (Conte Camerino et al., 1989; Satoh, 1998a). For example, taurine inhibits the inward rectifier K⁺ channel of isolated cardiomyocytes by reducing the channel open probability without affecting the single channel conductance (Satoh, 1998b). ATP-sensitive K⁺ (K_ATP) channels of cardiac and skeletal muscle fibers are also inhibited by the sulfonic amino acid. In fact, we have shown that taurine induces a reversible inhibition of the muscular K_ATP channel interacting with the sulfonylurea receptor (SUR) subunit without affecting the Ca²⁺ binding site (Tricarico et al., 2000a). In the case of voltage-dependent K⁺ channels, the action of taurine has been described to be Ca²⁺-dependent; in fact, the amino acid behaves as an agonist of the cardiac delayed rectifier K⁺ channel at low internal Ca²⁺ concentrations (10⁸ M) and as an antagonist in the presence of a high internal Ca²⁺ concentration (10⁶ M) (Satoh, 1998a).

The physiological significance of the observed inhibitory effects shown by taurine on different classes of K⁺ channels remains to be elucidated. One possibility is that taurine can serve to protect the fibers against ischemic insults. In fact, a release of the amino acid occurs during periods of ischemia reperfusion in cardiac fibers, and this would allow the opening of K_ATP channels and other K⁺ channels with rapid repolarization of the fibers and cytoprotective effects (Allo et al., 1997; Suleiman et al., 1997; Saransari and Oja, 1998; Tricarico et al., 2000a).

Although some of the therapeutic effects of this amino acid appear to involve the interaction of taurine with different classes of K⁺ channels, currently no data is available on the action of taurine on another type of K⁺ channel widely expressed in the tissues, including skeletal muscle, the Ca²⁺-activated K⁺ (K_Ca²⁺) channels. The opening of this type of channel, triggered by cellular depolarization and elevated intracellular Ca²⁺ ions in the excitable tissues, increases the duration of the hyperpolarization phase between bursts of action potentials, reducing the firing capability of the fibers (Hille, 1984; Tricarico et al., 1997). This helps to reduce the intracellular accumulation of Ca²⁺ ions occurring during bursts of action potentials in the fibers. However, persistent abnormal opening of muscle K_Ca²⁺ channels may also lead to the accumulation of K⁺ ions inside the t-tubule during muscle contraction, thus contributing to the phenomenon known as muscle fatigue.

In the present work, the effects of different concentrations of taurine applied "in vitro" on the intracellular face of patches excised from native skeletal muscle fibers were investigated using the patch-clamp technique on K_Ca²⁺ channels.

	Materials and Methods

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Muscle Preparations and Single Fiber Isolation. The flexor digitorum brevis (FDB) muscles were dissected from the rats under urethane anesthesia (1.2 g/kg). After dissection, the animals were rapidly killed with an overdose of urethane according to the Guide for Care and Use of Laboratory Animals prepared by the National Academy of Sciences (Washington, DC). Single muscle fibers were prepared from FDB muscles by enzymatic dissociation, as previously described (Tricarico and Conte Camerino, 1994).

Drugs and Solutions. The normal Ringer's solution contained 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 5 mM glucose, and 10 mM MOPS, pH = 7.2. The patch-pipette solution contained 150 mM KCl, 2 mM CaCl₂, and 10 mM MOPS, pH = 7.2. The bath solution contained 150 or 30 mM KCl, 5 mM EGTA, and 10 mM MOPS, pH = 7.2. CaCl₂ was added to the bath solution to give a free Ca²⁺ ion concentration of 8 µM or 16 µM. The calculations of the free Ca²⁺ ion concentration in the bath were performed as previously described (Tricarico et al., 1997, 2000b). Taurine was dissolved in the bath solution and tested in a range of concentrations from 10⁵ to 1.5¹ M. The possible influence of the osmolarity on K_Ca²⁺ channels was evaluated by applying a bath solution enriched with sucrose (20-100 mM) to the internal side of the excised patches. No significant effects were observed after the addition to the bath of a solution containing 20 to 100 mM concentrations of sucrose.

Patch Pipettes. The patch pipettes were prepared as previously described. Pipettes, having an average tip opening area of 5.7 ± 0.6 µm² (macropatches = 23), were used to measure the current sustained by multiple K_Ca²⁺ channels and their pharmacological properties. Moreover, the single channel conductance and the gating properties of the channels per unit area were measured using pipettes having a tip opening area of 0.9 ± 0.2 µm² (patches = 12).

Patch-Clamp Experiments. Experiments were performed in inside-out configurations using the standard patch-clamp technique. Recordings of macropatch K_Ca²⁺ currents were performed at 20°C, at different voltages (from 60 to +30 mV) of membrane potentials (V_m), for time periods of 300 to 400 s immediately after excision, with 150 mM KCl on both sides of the membrane, and in the presence of 8 or 16 µM concentrations of free Ca²⁺ ions in the bath solution (Tricarico et al., 1997, 2000b). Single channel recordings were performed at 20°C, at 60 mV or +30 mV of membrane potentials, for 120 to 200 s, and during voltage steps from 0 mV of holding potential to 70 and +70 mV (V_m). The macropatch currents and single channel currents were recorded at 1 kHz (filter = 0.2 kHz) and 20 kHz (filter = 2 kHz) of sampling rates, respectively, using an Axopatch-1D amplifier equipped with a CV-4 headstage (Axon Instruments, Foster City, CA).

Analysis of the Macropatch Current and Single Channel Current. The K_Ca²⁺ currents flowing through the macropatches excised from different FDB fibers were digitally averaged and were calculated by subtracting the baseline level of the currents from the open channel level, as previously described (Tricarico et al., 1997, 2000b). The baseline level for K_Ca²⁺ current was measured in the absence of free Ca²⁺ ions in the bath. The criteria for accepting the data entering within the digital average were based on the stability of the seal, the possible noises, and the presence of currents different from K_Ca²⁺ current. Macropatches containing voltage-gated K⁺ channels or inward rectifier K⁺ channels were excluded from the analysis. Currents from voltage-gated K⁺ channels or inward rectifier K⁺ channels were identified on the basis of their single channel conductance, voltage dependence, and lack of stimulation by Ca²⁺ ions. For example, the most common voltage-gated K⁺ channels active in our patches showed single conductance ranging between 10 and 19 pS and were identified in the absence of intracellular free Ca²⁺ ions during voltage steps going from 80 or 100 mV to depolarizing voltages, whereas inward rectifier K⁺ channels showed single channel conductance ranging between 10 and 40 pS and were identified in the absence of internal free Ca²⁺ ions at negative membrane potentials. K_ATP channels are inhibited by micromolar concentrations of Ca²⁺ ions (Tricarico et al., 2000a). The single channel current of K_Ca²⁺ channels was measured using the cursor method provided by the Fetchan program (Axon Instruments). The single channel conductance was calculated as the slope of the voltage-current relationship of the channel in the range of potentials from 70 to +70 mV. No correction for liquid-junction potential was made, and it was estimated to be <1 mV in our experimental conditions.

Statistics. The data are expressed as mean ± S.E. unless otherwise specified. The concentration-response relationship of K_Ca²⁺ currents versus drug concentrations constructed at a positive membrane potential of +30 mV and describing the antagonist action of taurine on K_Ca²⁺ channels fit the following equation: (I_drug  I_control)/I_control = E/(1 + ([Drug]/IC₅₀)ⁿ), whereas the concentration-response relationship of K_Ca²⁺ currents versus drug concentrations constructed at a negative membrane potential of 60 mV and describing the agonist action of taurine on K_Ca²⁺ channels fit the following equation: (I_drug  I_control)/I_control = E/(1 + (DE₅₀/[Drug])ⁿ), where (I_drug  I_control)/I_control is the ratio between the current measured in the presence of drug and that measured in the absence of drug; E is the observed effects (inhibition or activation) of the drugs on K_Ca²⁺ channels; DE₅₀ is the concentration of the drug needed to enhance the current by 50%; [Drug] is the concentration of the drug tested; n is the slope of the curves; and IC₅₀ is the concentration of the drug needed to reduce the currents by 50%. The algorithms of the fitting procedures used were based on the Marquardt least-squares fitting routine.

	Results

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The application of increasing concentrations of taurine to the macropatches excised from rat skeletal muscle fibers provoked a dose-dependent decrease in the muscle K_Ca²⁺ outward currents recorded at a positive membrane potential of +30 mV (V_m) in the presence of an 8 µM concentration of free Ca²⁺ ions in the bath. For example, the outward currents were decreased by 39 and 62% with 20 · 10³ and 60 · 10³ M concentrations of taurine, respectively (Fig. 1A). The IC₅₀ of taurine calculated using the fitting routine was 31.9 · 10³ ± 1 M (slope factor = 1.2) (n = 11 patches) (Fig. 1B).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effects of intracellular application of taurine on KCa2+ currents of rat skeletal muscle fibers. A, digital average of KCa2+ currents of 10 macropatches continuously recorded in inside-out configuration at different voltages in the presence of 150 mM KCl on both sides of the membrane and 8 µM free Ca2+ ions in the bath. At negative membrane potentials, the downward deflection of the current indicates channel opening, whereas at positive membrane potentials, the channel opening is indicated by the upward deflection of the current. The internal application of taurine enhanced the inward KCa2+ currents recorded at negative membrane potentials, while reducing the outward KCa2+ currents at positive membrane potentials. B, dose-response curve of normalized KCa2+ currents against taurine concentrations constructed at membrane potentials of +30 mV () and at 60 mV () in the presence of 150 mM KCl on both sides of the membrane and 8 µM free Ca2+ ions in the bath. At a membrane potential of +30 mV, a dose-dependent inhibition of the currents was observed following taurine application to the macropatches. In contrast, a membrane potential of 60 mV of taurine caused a dose-dependent activation of the currents.

In contrast, at negative membrane potentials in the presence of an 8 µM concentration of free Ca²⁺ ions in the bath, taurine exerted an opposite effect on muscle K_Ca²⁺ currents. In fact, 20 · 10³ and 60 · 10³ M concentrations of the sulfonic amino acid enhanced the inward K_Ca²⁺ current, increasing it by 56 and 67% at 40 mV (V_m), and 31 and 43% at 60 mV (V_m), respectively. The DE₅₀ of taurine calculated using the fitting routine was 46.7 · 10³ ± 2 M (slope factor = 1.3) (n = 9 patches) (Fig. 1B). In the absence of internal Ca²⁺ ions, taurine did not induce activation of K_Ca²⁺ currents at negative membrane potentials.

Single channel recordings revealed that the dualistic effects of taurine on K_Ca²⁺ currents observed at negative and positive membrane potentials were mainly due to changes in the reversal potential of K_Ca²⁺ channel to K⁺ ions (Fig. 2). This was demonstrated by taurine causing a dose-dependent rightward shift of the current-voltage relationship of the K_Ca²⁺ channel from 0.3 mV in the absence of taurine to +9.3 ± 2 and +16 ± 3 mV in the presence of 20 · 10³ and 60 · 10³ M concentrations of the sulfonic amino acid, respectively. Accordingly, 20 · 10³ and 60 · 10³ M concentrations of taurine reduced the single channel current at +30 mV (V_m) by 42 and 65%, while increasing it by 32 and 44% at 60 mV (V_m), respectively. The changes in the single channel currents observed following taurine application to the excised patches matched those observed in the macropatch currents.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of taurine on the current-voltage relationship of the KCa2+ channel of rat skeletal muscle fibers. The single channel currents were recorded in the presence of 150 mM KCl on both sides of the membrane and 8 µM free Ca2+ ions in the bath. The internal application of 20 · 103 M () or 60 · 103 M () concentrations of taurine caused a dose-dependent rightward shift of the current-voltage relationship of the KCa2+ channel to more positive potentials than the control () without affecting the single channel conductance.

No changes were observed in the single channel conductance following the in vitro application of 20 · 10³ and 60 · 10³ M concentrations of taurine to the patches containing K_Ca²⁺ channels. Indeed, the slope conductance calculated from the current-voltage relationships for the K_Ca²⁺ channel was 221 ± 12 pS (n patches = 8) in the controls and 228 ± 9 pS (n = 9 patches) and 222 ± 10 pS (n = 9 patches) in the presence of 20 · 10³ and 60 · 10³ M concentrations of taurine, respectively (Fig. 2).

Taurine did not affect the long-term gating properties of the K_Ca²⁺ channel. In fact, the application of a 30 · 10³ M concentration of the sulfonic amino acid to excised patches containing multiple K_Ca²⁺ channels did not alter the gating or the number of functional channels per patch area at positive or negative membrane potentials (Fig. 3, A-D). Furthermore, taurine did not alter the sensitivity of the K_Ca²⁺ channel to Ca²⁺ ions, which is demonstrated by the fact that the gating and the number of functional channels per patch area were unchanged after application of a 30 · 10³ M concentration of taurine to the patches at positive or negative membrane potentials.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effects of taurine on the long-term gating properties of the KCa2+ channel of rat skeletal muscle fibers. A, the open probability · number of functional channels plotted versus time was measured every 500 ms at a membrane potential of 60 mV in the presence of 8 or 16 µM concentrations of internal free Ca2+ ions. The internal application of 30 · 103 M concentration of taurine did not affect the gating properties of the channel or the Ca2+ sensitivity. B, single channel trace of the KCa2+ channel recorded at 60 mV of membrane potential in the presence of a 16 µM concentration of internal free Ca2+ ions and in the absence or presence of a 30 · 103 M concentration of taurine. A current from one active KCa2+ channel is represented. At this membrane potential, the sulfonic amino acid enhanced the single channel current without affecting the leak current. C, the open probability · number of functional channels plotted versus time was measured every 500 ms at a membrane potential of +30 mV in the presence of 8 or 16 µM concentrations of internal free Ca2+ ions. The internal application of a 30 · 103 M concentration of taurine did not affect the gating properties of the channel or the Ca2+ sensitivity. D, single channel trace of the KCa2+ channel recorded at +30 mV of membrane potential in the presence of a 16 µM concentration of internal free Ca2+ ions in the absence or presence of a 30 · 103 M concentration of taurine. Currents from two active KCa2+ channels are represented. At this membrane potential, the sulfonic amino acid reduced the single channel current without affecting the leak current.

In our experimental condition, taurine did not affect leak currents (Fig. 3, B and C). Concentrations of taurine lower than 5 · 10³ M did not alter the macroscopic or microscopic properties of the K_Ca²⁺ channel.

	Discussion

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We found that taurine applied on the intracellular face of the membrane patches excised from skeletal muscle fibers induced two opposite effects on sarcolemma K_Ca²⁺ currents. At positive membrane potentials, the outward K_Ca²⁺ currents decreased, whereas at negative membrane potentials, the sulfonic amino acid induced stimulation of inward K_Ca²⁺ currents. This is the result of the rightward shift of the reversal potential of the K_Ca²⁺ channel for K⁺ ions caused by taurine. Changes in the reversal potentials of ion channels may be related to modifications of the selectivity of the pore to K⁺ ions, to alteration of the surface charge potentials, and/or to changes in the local charge density (Hille, 1984). In our study, the observation that taurine altered the reversal potential of the K_Ca²⁺ channel for K⁺ ions without affecting the single channel conductance supports the hypothesis that the effects of the amino acid are related to changes in the surface charge potentials and/or change in the local charge density rather than to changes in the selectivity of the pore for K⁺ ions. It is possible that taurine affected the local charge density by binding to charge residues located at the mouth of the pore of the K_Ca²⁺ channel reducing the accessibility of K⁺ ions from the intracellular side of the membrane to the conduction pathway. In our experiments, the finding that the slope factor calculated by the fitting routine was about 1 for both the inhibition and activation of the K_Ca²⁺ channel by the sulfonic amino acid suggests that the stoichiometry of the interaction of taurine with the binding site is 1:1.

Similarly, a shift of the reversal potentials of the cardiac inward rectifier K⁺ channel and the embryonic Na⁺ channel toward more negative potentials following extracellular taurine application has also been described (Satoh, 1995, 1998b). This is not surprising considering that taurine is a charged amino acid capable of altering surface charge potentials by interacting with the polar phase of phospholipids. If the site(s) of interaction are located in the ion channel pore on residues critical for the conduction pathway, modification of the reversal potentials for the carried ion can be expected. Alternately, if the site of interaction of taurine is located in proximity to subunits critical for controlling channel activity and the pharmacological properties of the channel, changes in gating and in the response of the channel to drugs can be expected. This phenomenon occurs in the case of the action of taurine on muscle K_ATP channels in which the sulfonic amino acid induces a reversible inhibition of the muscular channel interacting with a site allosterically coupled to the SUR of the K_ATP channel complex at the interface between the membrane phospholipids and the SUR protein without altering the reversal potential for K⁺ ions (Tricarico et al., 2000a).

From a pharmacological point of view, taurine can therefore be considered an inhibitor of several classes of K⁺ channels, including Ca²⁺-activated K⁺ channels. In skeletal muscle, the capability of the sulfonic amino acid to reduce the outward K_Ca²⁺ current, even in the presence of the natural stimulatory ligand of the Ca²⁺ ions, may have important implications in those conditions associated with hyperkalemic states, such as ischemia-reperfusion and muscle fatigue, or in cases of an impairment of muscle contraction, such as the aging process (Pierno et al., 1998). The action of taurine, through inhibition of the outward currents carried by K_Ca²⁺ channels, would reduce the hyperpolarization phase following the firing of action potentials, thus controlling the Ca²⁺ homeostasis of the fibers and contributing to Ca²⁺-dependent physiological functions, such as muscle contraction. This corresponds with the fact that taurine supplementation shows therapeutic effects, improving the contractility functions of aged rat skeletal muscle (Pierno et al., 1998).

We believe that the agonist action shown by taurine at negative membrane potentials on inward currents of K_Ca²⁺ channels may have implications in those pathophysiological conditions associated with taurine depletion, such as ischemia-reperfusion in which a loss of taurine can favor the opening of K_Ca²⁺ channels and other K⁺ channels with fiber repolarization and cytoprotective effects (McPherson et al., 1993; Hearse, 1995; Han et al., 1996; Tricarico et al., 2000a). This corresponds with the observation that the loss of taurine from the tissues is considered as a protective mechanism against ischemia (Allo et al., 1997; Suleiman et al., 1997).