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Background and Purpose Despite the fact that chloride channels are involved in several physiological processes and acquired diseases, the availability of compounds selectively targeting CLC proteins is rather limited. ClC-1 channels are responsible for sarcolemma repolarization after an action potential in skeletal muscle fibers and have been associated with myotonia congenita and myotonic dystrophy as well as with other muscular physiopathological conditions. To date only a few ClC-1 blockers have been discovered, such as anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA), whereas no useful activator exists. The absence of a ClC-1 structure and the limited information regarding the binding pockets in CLC channels hamper the identification of improved modulators. Experimental Approach The aim of this study was to provide an in-depth characterization of drug binding pockets in ClC-1 through an integrated in silico and experimental approach. We searched putative cavities in a homology model of ClC-1 built upon an eukaryotic CLC crystal structure, and validated in silico data by measuring the blocking ability of 9-AC and NFA on mutant ClC-1 channels expressed in HEK 293 cells. Key Results We identified four putative binding cavities in ClC-1. 9-AC appears to interact with K231, R421 and F484 within the channel pore that are pivotal for ClC-1 block. We also identified one preferential binding cavity for NFA and propose R421 and F484 as critical residues. Conclusion and Implications This study represents the first effort to delineate, through a computational-functional protocol, the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins.

Background and Purpose Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database. Experimental Approach We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations. Key Results Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified. Conclusion and Implications These results suggest additional mechanisms of action for these sartans further to their primary AT1 receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands.

The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 μM. These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with KD values at −140 mV ranging from about 4 to 180 μM. In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket. Keywords: Skeletal muscle, CLC-1 chloride channel, chloride conductance, clofibric acid analogues Introduction CLC-1 is a voltage-dependent Cl− channel selectively expressed in skeletal muscle, where it is responsible for the maintenance of the membrane potential at rest and consequently for the electrical stability of the sarcolemma. Genetic defects of CLC-1 lead to inheritable myotonia, a disease characterised by abnormal membrane hyperexcitability and muscle stiffness, in mice, goats and humans (Steinmeyer et al., 1991; Koch et al., 1992). Both the small single-channel conductance (Pusch et al., 1994) and the probable localisation of the channel in the transverse tubules (Chua & Betz, 1991) do not allow the study of this muscle Cl− channel by direct inspection with the patch-clamp technique on native fibres. The measurement of the macroscopic Cl− conductance (gCl), the electrical parameter sustained by CLC-1, allows to gain insight into the activity and pharmacology of the Cl− channel in its native environment. The 2-(p-chlorophenoxy) propionic acid (CPP) and the 9-anthracenecarboxylic acid (9-AC) are two specific ligands of the CLC-1 channel (Jentsch et al., 2002; Pusch et al., 2002). Pioneering studies conducted in our laboratory on native skeletal muscle have demonstrated that CPP, having an asymmetric carbon atom α to the carboxylic group, is capable of stereoselectively modulating the native gCl (Conte Camerino et al., 1988; De Luca et al., 1992b). In particular, S(−)CPP blocks gCl in a concentration-dependent manner with a half-maximal concentration in the range of 10–20 μM, whereas the R(+) enantiomer produces a typical biphasic effect, enhancing gCl slightly at low concentrations (1–10 μM) and decreasing it at higher concentrations (>10 μM). The opposite effects produced by the individual enantiomers allowed us to hypothesise the presence of two different receptors or binding sites able, respectively, to enhance and decrease channel activity. The mechanism of the inhibition mediated by CPP has been studied in great detail using cellular lines or Xenopus oocytes as heterologous expression systems (Aromataris et al., 1999; Pusch et al., 2000). S(−)CPP blocks CLC-1 current in an apparently complex voltage-dependent manner and interferes with channel gating acting from the intracellular side. In particular, as demonstrated by single-channel recording of homologous CLC-0 chloride currents, S(−)CPP inhibits the individual protopores of the double-barrelled channel by binding to the pore preferentially when the channel is closed (Pusch et al., 2001). Furthermore, CPP is a specific ligand for the muscle CLC-1 channel, being much less effective on the ubiquitous CLC-2 channel and completely ineffective on renal CLC-5 and CLC-K channels from the extracellular side (Pusch et al., 2000; Liantonio et al., 2002). The phosphorylation of the CLC-1 protein by Ca-dependent protein kinase C (PKC) modulates channel activity in native skeletal muscle and in heterologous expression systems (De Luca et al., 1998; Rosenbohm et al., 1999). Furthermore, the sensitivity of CLC-1 to CPP enantiomers changes with the PKC-dependent phosphorylation state of the channel protein (De Luca et al., 1994; 1992a). These findings corroborate the idea that the measurement of gCl of native skeletal muscle fibres represents a useful system to obtain information about the modulation of CLC-1 activity, preserving the mechanisms of biochemical constituents that are fundamental for the channel physiology. On the other hand, taking into account the intracellular localisation of the hypothetical binding site of CPP (Pusch et al., 2000; 2001), inside-out patch-clamp recordings on heterologously expressed CLC-1 channel allow to study the interaction between the drugs and the amino-acid residues involved in the binding site independently from the capability of the molecule to cross the plasma membrane. The stereoselective effect of CPP and the reduced potency of clofibric acid and p-chlorophenoxyacetic acid (CPA), two achiral CPP-like compounds, in blocking native gCl (Conte Camerino et al., 1988; Loiodice et al., 1993), suggest that the asymmetric carbon atom of the drug molecule represents an important pharmacophore moiety. In order to determine the molecular requisites for the modulation of the native gCl and consequently the activity of CLC-1 channel, and with the goal of identifying high-affinity ligands, we performed a structure–activity relationship study by synthesising and evaluating the effects of a series of CPP derivatives (Figure 1). We initially screened the CPP-like compounds on the native chloride conductance of rat skeletal muscle using the two-microelectrode current-clamp technique. All tested derivatives resulted less potent with respect to CPP. Taking into account that membrane permeability can be a limiting step for the effect on gCl, the derivatives having physical–chemical properties different from that of CPP, as the phosphonate derivatives and bis-phenoxy derivatives (N.23 and N.25–33), were also tested on heterologously expressed CLC-1 performing inside-out patch-clamp recordings. This approach allowed to better define the interaction between drug and channel protein, corroborating a high-affinity interaction of the bis-phenoxy derivatives, in spite of their limited permeability. Figure 1 Chemical structures of different analogues of CPP. Methods Experimental procedure of muscle preparation Adult male Wistar rats of 350–400 g were used for the experiments. The electrophysiological experiments were made on isolated extensor digitorum longus (EDL) muscle. The muscle was removed under urethane anaesthesia (1.2 g kg−1, i.p.). Soon after the biopsy, the EDL muscle was stretched to about 1.5 times its resting length on a 3 mm plastic rod in a temperature-controlled muscle chamber at 30°C and perfused with a physiological solution (see below) in the absence and in the presence of tested compounds (De Luca et al., 1992b).

(1) Growth hormone secretagogues (GHS) exhibit potent growth hormone (GH)-releasing activity through the activation of a pituitary receptor. Here, we consider the possibility that GHS can target a specific receptor in rat skeletal muscle and have a role in the control of muscle function. (2) By means of the intracellular microelectrode technique, we found that in vitro application of hexarelin and L-163,255 dose dependently reduced resting chloride (gCl) and potassium (gK) conductances in rat skeletal muscle. These effects were prevented by the GHS-receptor antagonist [D-Lys-3]-GHRP-6, and by either phospholipase C or protein kinase C (PKC) inhibitors. Ghrelin, a natural ligand of GHS receptors, also induced a reduction of muscle gCl and gK, which was antagonised by [D-Lys-3]-GHRP-6. (3) Both GHS shifted the mechanical threshold for the contraction of muscle fibres towards more negative voltages. Accordingly, by means of FURA-2 fluorescent measurements, we demonstrated that L-163,255 induced a resting [Ca(2+)](i) increase, which was reversible and not blocked by nifedipine or removal of external Ca(2+). (4) Ageing is a condition characterised by a deficit of GH secretion, which in turn modifies the electrical and contractile properties of skeletal muscle. In contrast to GH, chronic treatment of aged rats with hexarelin or L-163,255 failed to restore the electrical and contractile muscle properties. Moreover, the two GHS applied in vitro were able to antagonise the beneficial effect on gCl and gK obtained through chronic treatment of aged animals with GH. (5) Thus, skeletal muscle expresses a specific GHS receptor able to decrease gCl and gK through a PKC-mediated intracellular pathway. This peripheral action may account for the lack of restoration of skeletal muscle function in long-term GHS-treated aged animals.

New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K+ channels (KATP) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl (–CH3), ethyl (–C2H5) or propyl (–C3H7) groups, while maintaining pharmacophore groups critical for conferring agonist properties. The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10−4M). In the presence of internal ATP, all the compounds increased the macropatch KATP currents. The order of potency of the molecules as agonists was −C3H7 (DE50=1.63 × 10−8M) >−C2H5 (DE50=1.11 × 10−7M)>–CH3 (DE50=2.81 × 10−7M)>cromak-slim (DE50= 1.42 × 10−5M). Bell-shaped dose–response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the KATP currents. The order of increasing potency as antagonists was cromakalim (IC50=1.15 × 10−8M)–CH3 (IC50=2.6 × 10−8M)>–C2H5 (IC50=4.4 × 10−8M)>–C3H7 (IC50=1.68 × 10−7M) derivatives. These results suggest that the newly synthesized molecules and cromakalim act on muscle KATP channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists. British Journal of Pharmacology (2003) 139, 255–262. doi:10.1038/sj.bjp.0705233

Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the proline derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized proline derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).

No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode ‘point’ voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8–12 weeks, during the active regeneration of hind limb muscles, and 6–8 months, when regeneration is complete. The EDL muscle fibres of 8–12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6–8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6–8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6–8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid. British Journal of Pharmacology (2001) 132, 1047–1054; doi:10.1038/sj.bjp.0703907

Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti-schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels. Using the patch-clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP-sensitive K(+) channel (K(ATP)) immediately after excision and on channels that underwent rundown. The cytoplasmic application of 20 mM of taurine reduced the K(ATP) current; this effect was reverted by washout of the drug solution. In this experimental condition the IC(50) was 20.1 mM. After rundown, taurine inhibited the K(ATP) current with similar efficacy. Competition experiments showed that taurine shifted the dose-response inhibition curve of glybenclamide to the left on the log-dose axis without significantly affecting those of ATP or Ca(2+) ion. Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration. Our data indicate that taurine inhibits the muscular K(ATP) channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of K(ATP) channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of K(ATP) channels and salvage the intracellular ATP content.

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more eAcient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. EAects of Me5 on Na + channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 mM) reduced the maximal sodium current (INa) by 29.7+4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+4.4 % (n=6) at 1 Hz. At same concentration (10 mM), Mex was incapable of producing any eAect (n=3). Me5 also shifted the steady-state inactivation curves by 77.9+0.9 mV (n=6) at 0.3 Hz and 712.2+1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 mM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The eAects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na + channel activity shows that Me5 did not aAect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.

1. The effects of mexiletine were evaluated on the ATP-sensitive K+ channel (K(ATP)) of rat skeletal muscle fibres using patch clamp techniques. The effects of mexiletine were studied on macropatch currents 20 s (maximally activated), 8 min (early stage of rundown) and 15 min (late stage of rundown) after excision in the absence or in the presence of internal ADP (50-100 microM) or UDP (500 microM). In addition, the effects of mexiletine were tested on single channel. 2. In the absence of ADP and UDP, mexiletine inhibited the current through maximally activated channels with an IC50 of -5.58+/-0.3 M. Nucleoside diphosphates shifted the current versus mexiletine concentration relationship to the right on the log concentration axis. UDP (500 microM) was more efficacious than ADP (50-100 microM) in this effect. 3. At the early stage of rundown, the sensitivity of the channel to mexiletine was reduced and nucleoside diphosphates, particularly UDP, antagonized the effect of mexiletine. At the late stage of rundown, mexiletine did not affect the currents. 4. At the single channel level, 1 microM mexiletine reduced the mean burst duration by 63% and prolonged the arithmetic mean closed time intervals between the bursts of openings without altering the open time and closed time distributions. Mexiletine did not affect the single channel conductance. 5. These results show that in skeletal muscle, mexiletine is a state-dependent K(ATP) channel inhibitor which either acts through the nucleotide binding site or a site allosterically coupled to it.

1 In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (GCl) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP). 2 For this purpose GCl in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-β-phorbol 12,13 dibutyrate (4-β-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on GCl were then tested. 3 IGF-1 (3.3 nM) had no effect of GCl on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of GCl induced by 4-β-PDB. No effects of IGF-1 were observed on GCl lowered by the phosphatase inhibitor okadaic acid (0.25 μM). 4 Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5–5 μM), not very effective in control conditions, increased the GCl lowered by the phorbol ester, but not the GCl lowered by okadaic acid. 5 In the presence of 4-β-PDB, GCl was differently affected by the enantiomers of CPP. The S(−)-CPP was remarkably less potent in producing the concentration-dependent reduction of GCl, whereas the R(+)-CPP caused an increase of GCl at all the concentrations tested. 6 In conclusion, the PKC-induced lowering of GCl is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands. British Journal of Pharmacology (1998) 125, 477–482; doi:10.1038/sj.bjp.0702107

The effects of a 4-month daily treatment with recombinant human growth hormone (GH) (150 μg kg−1) to aged rats were evaluated on the passive and active membrane electrical properties of extensor digitorum longus (EDL) muscle fibres in vitro by means of a two intracellular microelectrode technique. Chronic GH treatment completely restored the diameter and the membrane capacitance of aged EDL muscle fibres and significantly lowered the membrane resistance towards the adult value. There was also an increase of the threshold current, a shortening of the latency and an increase of the amplitude of the action potential and a significant amelioration of the membrane firing capability. The effects were almost fully attributable to a significant 50% increase of resting conductance to chloride ions (GCl), although an observed restoration of potassium conductance and a possible effect on voltage-activated sodium channels could contribute to the effects. EDL muscle fibres of untreated aged rats showed a different pharmacological response to 2-(p-chlorophenoxy) propionic acid (CPP) enantiomers from that seen in adult rats; the S-(−) isomer was less potent in blocking GCl and the R-(+) isomer always increased GCl instead of producing the typical biphasic effect observed in adult fibres (an increase of GCl at 1–10 μM and a decrease at higher concentrations). The 4-month-GH-treated aged rats showed a pharmacological sensitivity to CPP enantiomers similar to that of adults. The in vitro application of insulin-like growth factor I (IGF-I), the peripheral mediator of GH, produced a significant and irreversible increase of GCl of EDL muscle of untreated aged rats, an effect not observed in adults. This effect was completely inhibited by preincubation with 0.5 μM okadaic acid, suggesting that the IGF-I receptor transduction pathway can act on the phosphorylation state of the chloride channel through a serine-threonine protein phosphatase. The results show that the skeletal muscle chloride channel is a target of the impairment of GH/IGF-I axis occurring in aged subjects. The acute and chronic effects observed on GCl of aged muscle fibres suggest that the GH/IGF-I stimuli act through a modulation of channel phosphorylation state and through the synthesis of ‘adult’-like type chloride channels.

(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.

1 New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K(+) channels (K(ATP)) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl (-CH(3)), ethyl (-C(2)H(5)) or propyl (-C(3)H(7)) groups, while maintaining pharmacophore groups critical for conferring agonist properties. 2 The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10(-4) M). In the presence of internal ATP, all the compounds increased the macropatch K(ATP) currents. The order of potency of the molecules as agonists was -C(3)H(7) (DE(50)=1.63 x 10(-8) M)-C(2)H(5) (DE(50)=1.11 x 10(-7) M)-CH(3) (DE(50)=2.81 x 10(-7) M)cromak-slim (DE(50)= 1.42 x 10(-5) M). Bell-shaped dose-response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. 3 In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the K(ATP) currents. The order of increasing potency as antagonists was cromakalim (IC(50)=1.15 x 10(-8) M)or =-CH(3) (IC(50)=2.6 x 10(-8) M)-C(2)H(5) (IC(50)=4.4 x 10(-8) M)-C(3)H(7) (IC(50)=1.68 x 10(-7) M) derivatives. 4 These results suggest that the newly synthesized molecules and cromakalim act on muscle K(ATP) channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists.