The sarcolemma maintains the interior of the fiber at a negative potential (the membrane potential) when compared to the extracellular fluid while the fiber is in a resting state.
From: Equine Exercise Physiology, 2008
The sarcolemma maintains the intracellular milieu, actively transports substrates into the muscle cell, serves as a docking location for proteins originating in the basement membrane and cytoskeleton, and also transmits neural excitatory impulses that lead to muscle contraction. Facilitated diffusion of glucose across the sarcolemma occurs via glucose transporters (GLUT). GLUT-1 is constitutively present in the sarcolemma and provides basal amounts of glucose uptake, whereas GLUT-4 is present in the endosomes in the sarcoplasma, which migrate to and then dock and fuse with the sarcolemma when stimulated by insulin and contraction-dependent processes. Long-chain fats are transported across the sarcolemma by fatty acid translocase.
The sarcolemmal properties of excitation and conduction are largely due to the presence of membrane-spanning ion conducting pathways and channel gates within the sarcolemmal lipid bilayer that regulate the selective and nonselective conductance of sodium, potassium, calcium, and chloride. They activate (open) in response to ligands, transmitters, or changes in voltage and inactivate (close) by intrinsic regulatory processes. Voltage-gated channels contain additional voltage-sensing transmembrane domains and are essential for the generation and modification of action potentials. Ligand-gated ion channels are essential for setting myoplasmic calcium concentrations and establishing signal transduction pathways.
The sarcolemma forms tubular invaginations, t-tubules, at regular intervals along its length which traverse perpendicularly across the myofibrils at the junction of the A-bands and I-bands (Figure 12-3). The t-tubule membranes have a lower protein content but similar lipid content as the sarcolemma and contain numerous voltage-gated calcium channels called dihydropyridine receptors (DHPR). The t-tubules serve to transmit electrical impulses into the interior of the muscle fiber, where, through association with the intracellular membranous system, they can almost simultaneously initiate myofibrillar contraction.
Sarcoplasmic reticulum. The SR is an intracellular membrane-bounded compartment comprised of the terminal cisternae and longitudinal and corbular SR (Figure 3). The free walls of the terminal cisternae contain the RyR2 receptors and are apposed to the walls of the T-tubules, forming the dyadic cleft. Longitudinal SR contains the SR Ca2+-ATPase proteins, SERCA2, and its associated phosphoprotein inhibitor, phospholamban. Phosphorylation of phospholamban (by either protein kinase A or calcium–calmodulin kinase II) relieves the inhibition of SERCA2 and results in enhanced calcium uptake (Kranias, 1985). SR calcium is transported from the tubular lumen of the SR to the terminal cisternae, where it is stored mostly bound to calsequestrin, a low-affinity, high-capacity, calcium-binding protein (Bers QQ).
Figure 3. Electron micrograph and schematic diagram of a sarcomere. The darkly staining regions that flank the sarcomere are the Z lines. Myosin-containing thick filaments are in the center of the sarcomere and interact with actin-containing thin filaments by way of myosin heads that protrude from the thick filaments. Thin-filament regulatory proteins, the troponin and tropomyosin, provide calcium regulation of the actin–myosin interface. Thin filaments are anchored to the Z line, which is enriched in proteins such as α-actinin and CapZ (bottom) membrane complexes that concentrate over Z lines. The dystrophin–glycoprotein complex.
Myofilaments. Myofilaments comprise the contractile machinery of the cell and occupy 45–60% of the ventricular myocyte volume (Figure 3). The fundamental unit of the myofilament is the sarcomere, bounded by Z lines on each end, from which the thin actin filaments extend toward the center. At the center of the sarcomere is the M line, where the thick filaments are interconnected by M protein and myosin. Titin runs from the M line to the Z line in association with myosin and myosin-binding protein C. It acts as a scaffold for myosin deposition, stabilizes the thick filament, functions as a molecular spring, and plays a critical role in determining the passive stiffness of the heart (Brady, 1991). The Z lines are the anchoring site for cytoskeletal intermediate filaments and actin filaments at the intercalated disks and at focal adhesions between cells. The two major structural complexes involved in the connections between sarcomeric proteins and the extracellular matrix include the membrane-spanning integrin complex and the dystrophin complex.
Myosin. The myosin molecule consists of two heavy chains with a globular head, a long α-helical tail, and four myosin light chains (Figure 4). The myosin head forms cross-bridges with the thin actin filament through an actin-binding domain. The myosin head contains the myosin ATPase, which is responsible for the transduction of chemical to mechanical energy and work. Myosin heavy chain exists as two isoforms, α (fast ATPase and cross-bridge formation) and β (slow ATPase and cross-bridge formation). In higher mammals, including humans, the β-myosin isoform is predominant in the ventricles, whereas the α-isoform predominates in the atrium. The most accepted model of energy transduction is the sliding filament theory based on the formation and dissociation of cross-bridges between the myosin head and the thin filament that transits through different energetic states (Brenner, 1987). Two myosin light chains are associated with each myosin head and confer stability to the thick filament.
Thin filaments. The backbone of the thin filament is a helical double-stranded actin. Tropomyosin is a long flexible double-stranded, largely α-helical protein that lies in the groove between the actin strands and inhibits the interaction between actin and myosin. The troponin complex is comprised of a calcium-binding subunit, troponin C (TnC), an inhibitory subunit that binds to actin, troponin I (TnI), and a tropomyosin-binding subunit, troponin T, which is attached to tropomyosin. In the resting state, when
Figure 5. A mechanical model of the cross-bridge cycle. (a) Detached cross-bridge. (b) Cross-bridge prior to developing force. (c) Attached cross-bridge developing force stored in the elastic component. (d) Cross-bridge rotated and translated so that the filaments slide relative to one another. Each step in the cycle can be related to energetically different chemical states.
Mitochondria. Mitochondria comprise approximately 35% of ventricular myocyte volume and according to their cellular location they are designated as either subsarcolemmal or interfibrillar. Mitochondria are the site of oxidative phosphorylation and ATP generation. Although they have the capacity to buffer large amounts of Ca2+ and are a potential source of activator calcium, the classical teaching that their contribution to ECC is minimal in view of the short time constants involved has been challenged.
T.T. Hong, R.M. Shaw, in Ion Channels in Health and Disease, 2016
The sarcolemma is a lipid bilayer that is not uniform but rather separated into distinct membrane microdomains. Caveolae “little caves” exist in both T-tubule and lateral sarcolemma of ventricular cardiomyocytes. A caveolae is a flask-shaped structure enriched with cholesterol and sphingolipids formed by the cholesterol-binding scaffolding protein Caveolin-3 (Cav-3). Biochemical fractionation and electron microscopy studies have identified a subpopulation of many ion channels at caveolae, and loss of caveolae is associated with arrhythmogenesis.24 The precise role of caveolae on ion channel regulation and its significance still awaits further investigation. It has been reported that a subset of Cav1.2 channels is localized within caveolae, regulating calcium signaling.24 The mechanisms effecting ion channel enrichment at caveolae are unknown, but close interactions between caveolae and the cytoskeleton present an appealing possibility of targeted ion channel delivery to these sarcolemmal microdomains.25
We identified a new subdomain within cardiac T-tubules, the T-tubule minifolds sculpted by a membrane curvature formation protein BIN1.6 BIN1-induced T-tubule folds regulate extracellular calcium diffusion, affecting ion channel activity at the T-tubule surface. In addition to folds formation, BIN1 protein also facilitates microtubule-dependent targeting of Cav1.2 channels to T-tubules.26 Thus, by regulating Cav1.2 channels, BIN1 formed T-tubule folds may have an important role in excitation–contraction coupling.
Brian R. Berridge, .. Eugene Herman, in Fundamentals of Toxicologic Pathology (Third Edition), 2018
Cell Membrane Alterations
The sarcolemma represents an important cellular component that is usually exposed to the highest concentration of a substance. Some xenobiotics may change the electrical properties of the membrane. Muscle weakness, which occurs when concentrations of potassium are reduced, is apparently the result of a decrease in membrane excitability. Severe hypokalemia may lead to muscle fiber necrosis. In contrast, increased membrane excitability is the likely mechanism by which a number of agents (lithium, cimetidine, salbutamol, danazol, and captopril) cause muscle cramping.
The use of lipid-lowering drugs (statins and fibrates) may elicit a myopathy in both clinical and experimental situations. The precise mechanism by which these types of agents induce muscle fiber necrosis has not been completely elucidated, but there is evidence that the function of certain sarcolemmal ion channels is affected. Ion channel alterations may occur because these drugs inhibit the biosynthesis of important cholesterol-related membrane components. In addition, the lipid-lowering drugs also have important intracellular effects, which likely contribute to the muscle toxicity.
The hypocholesterolemic agent 20,25-diazacholesterol (no longer in clinical use) interferes with the biosynthesis of cholesterol and, as a result, causes accumulation of the cholesterol precursor desmosterol in the serum, the sarcolemma, and the SR. The presence of excessive amounts of desmosterol in the sarcolemma leads to excessive chloride permeability and myotonia (i.e., inability to relax voluntary muscles). Myotonia also occurs following exposure to the herbicidal compound, 2,4-dichlorophenoxyacetic acid (2,4-D). This agent causes metabolic alterations by inhibiting glucose-6-phosphate dehydrogenase, an effect that leads to membrane and subcellular changes; alterations in ion transport are also provoked. Advanced lesions induced by this agent include vacuolization and muscle fiber necrosis.
Alcohol consumption has been associated with skeletal muscle myopathy. Many pathogenic mechanisms are thought to be responsible for this toxicity, one of which appears to involve alterations in membrane control of electrolyte homeostasis.
Monocarboxylic acids, capable of inhibiting chloride conductance, also produce myotonia. Monensin, a carboxylic acid ionophore that selectively interacts with the voltage-dependent sodium (Nav) channel, consistently causes both skeletal and cardiac muscle necrosis in a variety of animals. Evidence suggests that these effects are due to calcium overloading in myofibers.
Injection of Type A Clostridium perfringens toxin (CPA) induces a unique myopathy. This toxin initially causes alterations in the sarcolemma. Subsequently, changes occur in the mitochondria and SR, and ultimately lesions are found in certain contractile components (I and Z bands, A-band filaments). These changes arise through the phospholipase activity of CPA, which breaks down cellular membranes. Clostridium chauvoei toxin A (CctA) is a potent cytotoxin that is a key hemolytic and necrotizing component responsible for inducing “blackleg,” an infectious hemorrhagic necrosis affecting the hind quarters of cattle (Figure 10.9) and other ruminants.
Figure 10.9. Hemorrhagic necrosis (blackleg) of the skeletal muscles in the hind quarters of a steer is indicated by multifocal dark red foci with emphysema (i.e., gas bubbles). Tissue sections demonstrated acute myofiber necrosis, shown by loss of striations and nuclei (*), in association with interstitial expansion by edema fluid (clear space with flocculent eosinophilic protein) and hemorrhage. The causative bacillus, Clostridium chauvoei, also is visible in the interstitium (arrows).Source
José-Luis L. Rivero, Richard J. Piercy, in Equine Sports Medicine and Surgery (Second Edition), 2014
Electrical and ionic properties of the sarcolemma
The sarcolemma maintains the interior of the fiber at a negative potential (the membrane potential) when compared to the extracellular fluid while the fiber is in a resting state. The negative potential is derived from the disequilibrium of ionic concentrations (mostly Na+ and K+) across the membrane and is generated partly by the action of the Na+/K+ ATPase pump, which extrudes three Na+ ions for every two K+ ions taken up. This results in the cytoplasm having a much higher K+ concentration but much lower Na+ concentration than the extracellular fluid. The remainder of the membrane potential is derived from the tendency of ions to diffuse down their electrochemical gradients across the semipermeable membrane.
Acetylcholine released from pre-synaptic nerve terminals at neuromuscular junctions (end-plates; Fig. 6.11B) binds to acetylcholine receptors and increases the conductance of the post-junctional membrane to Na+ and K+. The inward movement of Na+ down its concentration gradient predominates, causing a transient depolarization (about 20 mV) in the end-plate, known as the end-plate potential. This depolarization is sufficient to activate sarcolemmal voltage-gated Na+ channels – the channels that are mutated in hyperkalemic periodic paralysis. These Na+ channels open, eliciting propagation of an action potential along the membrane. Following this, and in response to depolarization, voltage-gated K+ channels open, resulting in the downswing of the action potential. The action potential therefore conducts rapidly along the sarcolemma in a wavelike fashion away from the neuromuscular junction.
G.T. Carter, in Encyclopedia of the Neurological Sciences (Second Edition), 2014
Muscle Membrane (Sarcolemma) Disorders
Impairment of sarcolemma excitability due to K+-mediated depolarization of the contracting fibers may result in excessive fatigue in myotonia congenita (MC), type 1 myotonic dystrophy (MD), hyperkalemic familial periodic paralysis (HKFPP), and paramyotonia congenita. In hereditary myotonic disorders (MC and MD), the fatigue appears within seconds of voluntary or stimulated contractions, but the paresis gradually wears off (warm-up phenomenon) as the exercise continues. The abnormal genotype expression of Cl– channel in MC and possible abnormality of Na+ channel in MD result in impairment of excitability of sarcolemma. However, in MD patients, in addition to impairment of sarcolemma, both mitochondrial and glycogenolytic functions are affected and may contribute to fatigue and weakness. In HKFPP and paramyotonia congenita (with genetic abnormality of Na+ channels), fatigue occurs less rapidly (in minutes) and tends to become worse after the voluntary contractions cease than in myotonic disorders (seconds).
Holly A. Shiels, in Fish Physiology, 2017
2.1.2 Electrical Connections Between Cells
The SL also contains gap junctions that connect cardiomyocytes electrically. In fish, as in other vertebrate hearts, gap junctions are formed by two hemichannels of connexon from opposing cells that come together to form a large conductance pore (a connexin). These form a low-resistance electrical pathway that allows for the passage of ions, signaling molecules, and metabolites between cells (Beyer et al., 1988; Severs et al., 2008). In mammals, approximately 1000 connexins are required to form the gap junction (Bruzzone et al., 1996), which is concentrated in the intercalated discs, and thus, colocalized with both fascia adherens and desmosomes. The mammalian heart predominantly expresses Cx43, Cx40, and Cx45, with Cx43 being by far the most prominent (Boyett et al., 2006). The numeral in the name of each connexin refers to their molecular weight. The working myocytes of the ventricle and atrium are strongly interconnected by Cx43 gap junctions, whereas Cx40 and Cx45 are more strongly expressed in the conduction system, and Cx30 is important during development (Valiunas et al., 2000). Several functional differences have been observed between various connexin subtypes, including pore conductance, size selectivity, charge selectivity, voltage gating, and chemical gating (Campbell et al., 2014). It is the combination of these factors, and their specific distribution within cardiac tissue, that allow connexins to control conduction velocity and action potential propagation through the heart (Johnstone et al., 2009; Valiunas, 2002; Valiunas and Weingart, 2000; Valiunas et al., 2000).
Connexins are highly conserved between species, and although studies are still limited, similarities between fish and mammalian connexins have been documented in terms of structure, function, and regulation; however, the names can vary due to minor differences in molecular weight. As in mammals, fish connexins can form heterologous as well as homologous gap junctions (Bolamba et al., 2003). The expression pattern of Cx43 in the developing zebrafish heart is very similar to that in mammals (Chatterjee et al., 2005). In the adult zebrafish, Cx45.6 (analogous to mammalian Cx40) is present in the atrium and ventricle (Christie et al., 2004). Interestingly, the zebrafish mutant ftk exhibits a severe cardiac developmental phenotype related to a loss of function of Cx36.7 (Sultana et al., 2008), and disrupting Cx48.5 expression results in severe cardiovascular deficiencies in embryonic and adult zebrafish (Cheng et al., 2004). Moreover, zebrafish studies have shown that a Cx46 mutation in the heart not only leads to disturbed electrical conductance in the ventricle, but this conduction impairment can also induce cardiac remodeling (Chi et al., 2010). Thus, information on the role of connexins in developmental patterning, and to a lesser extent in activation sequences, is limited in fish and exclusive to zebrafish hearts (Jensen et al., 2013). Currently, nothing is known about how variable expression and the regulation of connexins affect the conduction velocity between myocytes in other fishes.
The SL membrane also contains the ion channels and transporters responsible for ion flow during EC coupling, as illustrated in Fig. 2 and discussed in detail later.
The sarcolemma is characterized by a larger resting permeability for Cl− (gCl) than for K+ (gK). The evidence that Cl− permeation occurs through specific channels was soon derived from pharmacology, as it could be specifically blocked by inorganic (e.g., external Zn2+) and organic molecules such as 9-anthracene-carboxylic acid (9-AC) (Fig. 1). The main physiological role for the large gCl is to maintain the electrical stability of the sarcolemma. In fact in pioneering studies, Bryant showed that the hyperexcitability recorded in the intercostal muscle of myotonic “fainting” goat was related to an abnormally low gCl, and could be reproduced by 9-AC, putting the basis for the discovery of a large series of genetic diseases due to mutations in membrane ion channels (Bryant and Morales-Aguilera, 1971).
Since then, the physiological and pharmacological properties of muscle gCl were actively studied by classical two microelectrode current-clamp recordings and were pivotal for the future studies on cloned channel proteins (see following paragraphs). For instance, the evidence that gCl increases age-dependently in rat EDL muscle during the first month of postnatal life, contributed, along with its sensitivity to 9-AC, to support that the ClC-1 protein was indeed the channel accounting for the macroscopic resting conductance (Conte Camerino et al., 1989b; Steinmeyer et al., 1991).
Other than 9-AC and the agents classically defined as “Cl− channel blockers,” as for example 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and diphenylamine-2-carboxylate (DPC), other drugs can affect gCl (Camerino et al., 1989) (Table I). The main finding for the identification of specific ClC-1 modulators was the observation that a hypolipidemic drug, clofibrate, was able to induce an “iatrogen” form of myotonia. Apart from an unspecific membrane effect, likely due to change in the lipid environment, it was rapidly demonstrated that clofibric acid, the active in vivo metabolite of clofibrate, could specifically block muscle gCl, in a concentration-dependent manner, when applied in vitro (Conte-Camerino et al., 1984). This discovery opened the way toward an intense study of structure-activity relationship using a large number of clofibric acid derivatives, which turned out to be important pharmacological tools for studying various members of the CLC channel family (Pusch et al., 2000) (see following paragraphs). The 2-p-chlorophenoxy propionic acid (CPP) (Fig. 1), a chiral molecule, allowed also to investigate the possible stereoselectivity of the drug-binding site on muscle Cl− channels. In the native environment, the two enantiomers showed an opposite behavior. S(−)-CPP blocks gCl concentration-dependently and is one of the most potent compounds with an IC50 of about 15 μM. R(+)-CPP is much less potent in blocking gCl, but shows at low concentrations (1–5 μM) the ability to increase gCl (Conte-Camerino et al., 1988). This behavior was well fitted with a model of two sites able to oppositely modulate gCl and on which the enantiomers can act with different affinity and intrinsic activity (De Luca et al., 1992). The “opener” activity of R(+)-CPP is not observed for ClC-1 expressed in heterologous systems, suggesting that for the native muscle Cl− channel, some aspect of the native tissue plays an important role for modulating drug sensitivity (Aromataris et al., 1999; Pusch et al., 2000). However, it is worth noticing that a similar hypothesis, that is, the presence of both an “agonist” and an “antagonist” site, is now proposed for the renal CLC-K channels (Liantonio et al., 2006b).
|Clofibric acid derivatives||Direct high-affinity interaction||Block of gCl. The R(+) isomer can increase gCl at low concentrations||The effects are always detectable with differences that are age related||Blockers can induce an iatrogen-myotonia|
|Taurine||Low-affinity interaction (exogenous)||Increase of gCl||The effects are more evident on fast than on slow muscle types and in condition of taurine depletion||Taurine supplementation can restore gCl in aged and dystrophic muscles|
|Phorbol esters||Activation of PKC||Block of gCl||The effects are always detectable, with differences that are age related or fiber-phenotype dependent||Overactivity can lead to a myotonic state|
|IGF-1||Activation of a phosphatase||Increase in gCl||The effects are more evident in condition of PKC overactivity or in slow-twitch fibers||IGF-1 has proved beneficial effect in aging and dystrophic conditions|
|GH||IGF-1-mediated activation of phosphatase?||Increase in gCl||The effects are more evident in aged subjects||GH has proved beneficial effects during aging|
|Ghrelin||Receptor-mediated activation of PKC||Reduction of gCl|
|Statins||Direct or indirect (cholesterol pathways or PKC mediated)?||Reduction of gCl||The reduction of gCl may contribute to myophaties by statins|
|Niflumic acid||Both direct and PKC mediated||Reduction of gCl||The reduction of gCl may account for possible side effects by NSAID on muscle function on chronic use|
The extensive structure–activity studies allowed to gain insight into the molecular requisites for modulating gCl, and, therefore, for drug–channel interactions. Structure modifications were conducted in all parts of the CPP molecule potentially involved in binding such as the chiral center, the aromatic moiety, the acid function, and the oxygen atom of the aryloxy group. It was demonstrated that CPP is the most active structure on muscle gCl and that—other than the chiral center—a pivotal role is played by the carboxylic function, ensuring a proper acidity, the halogens on the aromatic ring, ensuring the proper electronic clouds, and the oxygen nearby the aromatic ring (Liantonio et al., 2003). Based on experiments with cloned ClC-1, it could be shown that the binding site for CPP and derivatives is directly accessible only from the intracellular side (Pusch et al., 2000). Thus, assaying drug efficacy in intact skeletal muscle fibers bears the complication that the drug has to enter the cytoplasm (see below).
Muscle gCl is a highly sensitive index of muscle function, being generally one of the first parameters to be changed in many pathophysiological conditions, such as aging, denervation, and dystrophic degeneration, as possible consequence of changes in channel expression and/or function (Conte Camerino et al., 1989b; De Luca et al., 1990; Pierno et al., 1999; De Luca et al., 2003). Consequently, gCl can be directly or indirectly sensitive to the action of various pharmacologically modulated pathways.
For instance, muscle gCl is controlled by biochemical pathways involving a system of protein kinases and insulin‐like growth factor‐1 (IGF-1)-sensitive phosphatases. A phorbol ester-sensitive protein kinase C (PKC) can potently block gCl and the phosphorylation state may control the trafficking of ClC-1 to the sarcolemma, its expression in physiological conditions as well as its drug sensitivity (De Luca et al., 1994, 1998; Rosenbohm et al., 1999; Papponen et al., 2005). Nonetheless, such a mechanism can also play a role in the phenotypic-dependent difference in gCl between fast-twitch and slow-twitch muscles, as well as in its modulation in conditions as disuse and microgravity in which muscle plasticity is activated (Pierno et al., 2002; Desaphy et al., 2005). Interestingly, even growth hormone, likely through production of IGF-1, or ghrelin, through a direct modulation of a muscular receptor, can increase or decrease gCl, respectively, by acting through the biochemical modulatory pathways (De Luca et al., 1997; Pierno et al., 2003). As these latter require the native environment, their influence on the effect of direct channel modulators is not easy to study on heterologously expressed channels.
Another interesting modulator of ClC-1 is taurine, an osmolyte usually present in high concentrations in skeletal muscle. Pharmacological and structure–activity relationship studies support the ability of taurine to control gCl, acting on a low-affinity site (mM range) nearby the channel (Pierno et al., 1994). The main activity of taurine is to increase gCl. Preliminary two microelectrode voltage-clamp recordings showed that in vitro application of taurine modestly enhances the Cl− currents sustained by human ClC-1 heterologously expressed in Xenopus oocytes. In parallel, taurine slightly shifts the channel activation toward more negative potentials, an effect that possibly accounts for the increase in resting gCl observed in native fibers during current-clamp recordings (Conte Camerino et al., 2004). The low-affinity site may account for taurine effectiveness in some forms of myotonic states (Conte Camerino et al., 1989a).
Other than a pharmacological action, taurine can also exert a long-term physiological control on the function of muscle Cl− channels. In fact, a depletion of taurine content decreases gCl; this effect may be due to the ability of taurine to modulate the pathways (Ca homeostasis, kinase/phosphatase pathways) involved in the maintenance of ClC-1 in an active state (De Luca et al., 1996). Accordingly, the in vivo treatment with taurine, likely acting by restoring intracellular pools, may counteract the gCl impairment due to diseases, such as muscular dystrophy, or to physiological states as aging (Pierno et al., 1999; De Luca et al., 2003).
On the other hand, drugs with side effects on skeletal muscle can have gCl as a first target. For instance, statins with a lipophilic structure can reduce muscle gCl (Pierno et al., 1995), a cellular event that may account for some of the muscle effects described for this class of therapeutic compounds. Although the mechanism by which statins can act on Cl− channels is under investigation, possible hypotheses include the reduction of cholesterol synthesis and consequently the alteration of cholesterol-dependent pathways, as well as the drug activity on the biochemical events involved in ClC-1 modulation. Interestingly, even niflumic acid (NFA), a drug belonging to nonsteroidal anti-inflammatory drugs (NSAIDs), has been found to decrease muscle gCl both directly and through a PKC-mediated action due to the mobilization of intracellular Ca (Liantonio et al., 2006a). Also in this case, the mechanism can lead to unwanted muscular effects on chronic use of the drug. The indirect modulation of Cl− channels by drugs able to affect or rather improve skeletal muscle function may seem far from the direct action of specific tools, as CPP derivatives. Nonetheless, these lines of evidence suggest that ClC-1, and possibly other members of the CLC family, may undergo a strict control through not yet defined pathways, subunits, or enzymatic systems able to affect, in the native environment, the biophysical and pharmacological properties of the channel.