Voltage-Dependent Antagonist/Agonist Actions of Taurine on Ca²⁺-Activated Potassium Channels of Rat Skeletal Muscle Fibers

Materials and Methods

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 10⁻⁵ to 1.5⁻¹ 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).

Different concentrations of taurine were applied on the intracellular side of the macropatches and micropatches, and their effects were tested on macropatch K_Ca²⁺ currents and on single channel currents during voltage steps and at constant voltages with 150 mM KCl on both sides of the membrane in the presence of 8 and 16 μM concentrations of free Ca²⁺ ions in the bath. Before recording, the patches were exposed to taurine for about 20 s.

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.

The effects of taurine on the gating properties of single K_Ca²⁺ channel were expressed as the open probability · the number of functional channels (N · P_open). The long-term gating properties and the Ca²⁺sensitivity of the channels in the presence or absence of taurine were investigated by plotting the N ·P_open measured every 500 ms against time at different voltages in the presence of different concentrations of internal Ca²⁺ ions (Tricarico et al., 1997).

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_controlis 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

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 · 10⁻³ and 60 · 10⁻³ M concentrations of taurine, respectively (Fig. 1A). The IC₅₀of taurine calculated using the fitting routine was 31.9 · 10⁻³ ± 1 M (slope factor = 1.2) (n = 11 patches) (Fig. 1B).

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 · 10⁻³ and 60 · 10⁻³ 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 · 10⁻³± 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 · 10⁻³ and 60 · 10⁻³ M concentrations of the sulfonic amino acid, respectively. Accordingly, 20 · 10⁻³ and 60 · 10⁻³M 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.

No changes were observed in the single channel conductance following the in vitro application of 20 · 10⁻³ and 60 · 10⁻³ 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 (npatches = 8) in the controls and 228 ± 9 pS (n = 9 patches) and 222 ± 10 pS (n = 9 patches) in the presence of 20 · 10⁻³ and 60 · 10⁻³ 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 · 10⁻³ 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 · 10⁻³ M concentration of taurine to the patches at positive or negative membrane potentials.

In our experimental condition, taurine did not affect leak currents (Fig. 3, B and C). Concentrations of taurine lower than 5 · 10⁻³ M did not alter the macroscopic or microscopic properties of the K_Ca²⁺ channel.

Discussion

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).