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The regulation of pH across the t-system membrane of skeletal muscle fibers is poorly understood. Using a sealed tubule preparation, Launikonis et al. reveal Na+/H+ exchange activity and characterize the properties of the diffusional and NHE proton fluxes across the t-system., The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system’s role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system’s role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

Vascular smooth muscle tone can be regulated by angiotensin II, which enhances TRPV4 channel activity via AKAP150-bound protein kinase C. Tajada et al. show that the effect of AKAP150 on TRPV4 channels is inversely proportional to the distance between them, which varies with sex and arterial bed., TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as “TRPV4 sparklets.” TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα.

Demonbreun et al. visualized muscle membrane repair in real time after laser-induced microdamage. Annexin proteins were observed to form a repair cap at the site of injury, supporting a shoulder-like structure containing EHD1, EHD2, dysferlin, and MG53., Disruption of the plasma membrane often accompanies cellular injury, and in muscle, plasma membrane resealing is essential for efficient recovery from injury. Muscle contraction, especially of lengthened muscle, disrupts the sarcolemma. To define the molecular machinery that directs repair, we applied laser wounding to live mammalian myofibers and assessed translocation of fluorescently tagged proteins using high-resolution microscopy. Within seconds of membrane disruption, annexins A1, A2, A5, and A6 formed a tight repair “cap.” Actin was recruited to the site of damage, and annexin A6 cap formation was both actin dependent and Ca2+ regulated. Repair proteins, including dysferlin, EHD1, EHD2, MG53, and BIN1, localized adjacent to the repair cap in a “shoulder” region enriched with phosphatidlyserine. Dye influx into muscle fibers lacking both dysferlin and the related protein myoferlin was substantially greater than control or individual null muscle fibers, underscoring the importance of shoulder-localized proteins. These data define the cap and shoulder as subdomains within the repair complex accumulating distinct and nonoverlapping components.

Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart.

Skeletal muscle activation requires action potential (AP) initiation followed by its sarcolemmal propagation and tubular excitation to trigger Ca(2+) release and contraction. Recent studies demonstrate that ion channels underlying the resting membrane conductance (G(M)) of fast-twitch mammalian muscle fibers are highly regulated during muscle activity. Thus, onset of activity reduces G(M), whereas prolonged activity can markedly elevate G(M). Although these observations implicate G(M) regulation in control of muscle excitability, classical theoretical studies in un-myelinated axons predict little influence of G(M) on membrane excitability. However, surface membrane morphologies differ markedly between un-myelinated axons and muscle fibers, predominantly because of the tubular (t)-system of muscle fibers. This study develops a linear circuit model of mammalian muscle fiber and uses this to assess the role of subthreshold electrical properties, including G(M) changes during muscle activity, for AP initiation, AP propagation, and t-system excitation. Experimental observations of frequency-dependent length constant and membrane-phase properties in fast-twitch rat fibers could only be replicated by models that included t-system luminal resistances. Having quantified these resistances, the resulting models showed enhanced conduction velocity of passive current flow also implicating elevated AP propagation velocity. Furthermore, the resistances filter passive currents such that higher frequency current components would determine sarcolemma AP conduction velocity, whereas lower frequency components excite t-system APs. Because G(M) modulation affects only the low-frequency membrane impedance, the G(M) changes in active muscle would predominantly affect neuromuscular transmission and low-frequency t-system excitation while exerting little influence on the high-frequency process of sarcolemmal AP propagation. This physiological role of G(M) regulation was increased by high Cl(-) permeability, as in muscle endplate regions, and by increased extracellular [K(+)], as observed in working muscle. Thus, reduced G(M) at the onset of exercise would enhance t-system excitation and neuromuscular transmission, whereas elevated G(M) after sustained activity would inhibit these processes and thereby accentuate muscle fatigue.

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (I(Cl)) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward I(Cl), and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and I(Cl) acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be I(Cl) dependent since its magnitude varied in close correlation with the amplitude and time course of I(Cl). While the properties of I(Cl), and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (P(Cl)) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if P(Cl) was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded I(Cl) arises from TTS contributions.

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15-16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at -140 mV; -39.0 +/- 4.5 and -42.3 +/- 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V(1/2) was -61.0 +/- 1.7 and -64.5 +/- 2.8 mV; k was 20.5 ± 0.8 and 22.8 +/- 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 +/- 36 to 312 +/- 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an approximately 40 and 60% reduction in membrane capacitance in FDB fibers from 15-16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90 degrees out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein-tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.

In spite of the detailed understanding reached in the last 50 years about the molecular mechanisms of muscle excitability, the localization of the Cl− conductance, GCl, in muscle has remained highly contended. In this issue, [Lueck et al.][1] address this long-standing controversy, measuring

Disruption of dystrophin or the sarcoglycans within the dystrophin protein complex leads to muscular dystrophy. The dystrophin complex is enriched in myofibers and concentrated into costameres, rib-like structures in the plasma membrane. In the absence of dystrophin, the plasma membrane becomes

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

In smooth muscle, the gating of dihydropyridine-sensitive Ca(2+) channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca(2+) influx and determining the bulk average cytoplasmic Ca(2+) concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca(2+) signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca(2+) current (I(Ca)) and evoked a rise in [Ca(2+)] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca(2+)] increase ([Ca(2+)](c)) was approximately uniform, whereas that of the subplasma membrane space ([Ca(2+)](PM)) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca(2+) channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca(2+) channels were not normally constitutively active. The repetitive localized [Ca(2+)](PM) rises ("persistent Ca(2+) sparklets") that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca(2+) channels regulate the bulk average [Ca(2+)](c). A dihydropyridine blocker of Ca(2+) channels, nimodipine, which blocked I(Ca) and accompanying [Ca(2+)](c) rise, reduced neither the resting bulk average [Ca(2+)](c) (at -70 mV) nor the rise in [Ca(2+)](c), which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca(2+) channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca(2+)](c) regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca(2+)].

Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin–glycoprotein complex (DGC) from the sarcolemma. We show that sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophin-deficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin–glycoprotein complex (UGC) at the extrasynaptic membrane to compensate for the loss of dystrophin. Utrophin is normally restricted to the neuromuscular junction, where it replaces dystrophin to form a functionally analogous complex. SSPN directly interacts with the UGC and functions to stabilize utrophin protein without increasing utrophin transcription. These findings reveal the importance of protein stability in the prevention of muscular dystrophy and may impact the future design of therapeutics for muscular dystrophies.

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform–specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.

Disruption of the dystrophin–glycoprotein complex caused by genetic defects of dystrophin or sarcoglycans results in muscular dystrophy and/or cardiomyopathy in humans and animal models. However, the key early molecular events leading to myocyte degeneration remain elusive. Here, we observed that the growth factor–regulated channel (GRC), which belongs to the transient receptor potential channel family, is elevated in the sarcolemma of skeletal and/or cardiac muscle in dystrophic human patients and animal models deficient in dystrophin or δ-sarcoglycan. However, total cell GRC does not differ markedly between normal and dystrophic muscles. Analysis of the properties of myotubes prepared from δ-sarcoglycan–deficient BIO14.6 hamsters revealed that GRC is activated in response to myocyte stretch and is responsible for enhanced Ca2+ influx and resultant cell damage as measured by creatine phosphokinase efflux. We found that cell stretch increases GRC translocation to the sarcolemma, which requires entry of external Ca2+. Consistent with these findings, cardiac-specific expression of GRC in a transgenic mouse model produced cardiomyopathy due to Ca2+ overloading, with disease expression roughly parallel to sarcolemmal GRC levels. The results suggest that GRC is a key player in the pathogenesis of myocyte degeneration caused by dystrophin–glycoprotein complex disruption.

α-Dystrobrevin (DB), a cytoplasmic component of the dystrophin–glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking αDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main αDB isoforms, αDB1 and αDB2, with common NH2-terminal but distinct COOH-terminal domains. αDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. αDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in αDB−/− mice prevented muscle fiber degeneration; however, only αDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of αDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, αDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, αDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.

We read with interest the recent article by Lueck et al. (2010) and the related commentary by Zifarelli and Pusch (2010) and wish to challenge the conclusion reached from their published data that ClC-1 chloride channels in adult mammalian skeletal muscle fibers are located “within the sarcolemma and not the T-tubular system” (Lueck et al., 2010). First, the confocal imaging provided cannot be used as evidence that there are many more ClC-1 channels in the sarcolemma than in the T-tubular system. This is because the average diameter of the T-tubules is severalfold smaller ( 200 nm, set by confocal resolution), and the T-tubules are distributed throughout the volume of the fiber, whereas the surface membrane is concentrated in the same “line” in the X–Y plane. Therefore, one would need a considerably higher gain to visualize the ClC-1 channels in the T-tubules than in the sarcolemma, even if the ClC-1 channels were equally distributed between the sarcolemma and the T-tubules. Thus, the fact that ClC-1 can be seen at the surface membrane and not deeper in the fiber in Fig. 7 A is completely expected from the low gain image used. This is precisely what we have previously vividly shown in regard to caveolin-3, a protein that is often mistakenly said to be present only at the sarcolemma and not in the T-system. This protein in fact is present in very large amounts in the T-tubular system, which is readily apparent if the confocal section is examined at adequate photomultiplier gain (e.g., Fig. 6 in Murphy et al., 2009), whereas at lower gain, only the sarcolemmal caveolin-3 is apparent (see Fig. 9 in Murphy et al., 2009) in an image looking almost identical to that in Fig. 7 A in Lueck et al. (2010). Concerning the detubulation experiments shown in Fig. 1, we also query why the surface membrane staining by di-8-ANEPPS is reduced after detubulation (A, right) and whether the gain used allows adequate visualization of the T-system, particularly if it is dilated and distorted after the detubulation procedure. Second, Lueck et al. (2010) misinterpret their confocal images of GFP-tagged ClC-1 expression in muscle (Figs. 9 A and 10) as showing “robust sarcolemma expression and a low level of intracellular localization in some fibers.” The authors evidently base this on the relatively strong intensity fluorescence signal from the sarcolemma (see point above) compared with that in the fiber. For a correct interpretation of their data, they need to compare the total fluorescence signal from the sarcolemma with the total fluorescence signal from inside of the cell. From Figs. 9 A and 10 A, it appears that the total fluorescence signal from inside the cell is greater than the total fluorescence signal of the entire sarcolemma, thus contradicting the authors’ claim above and the related statement made in the accompanying commentary (Zifarelli and Pusch, 2010) that “the fluorescence signal from tagged ClC-1 was confined to the sarcolemma.” Furthermore, when the transverse section in Fig. 10 A is examined closely, it appears that the intracellular ClC-1 signal is organized in “rings” ∼1 µm in diameter, as would be expected if it originates from the T-tubules wrapped around individual myofibrils, very much like that seen with caveolin-3 (see Fig. 4 B in Murphy et al., 2009). We also find it rather puzzling that the authors discard the possibility that a substantial amount of GFP-tagged ClC-1 chloride channels is expressed in the T-tubules, considering that they cite the paper by DiFranco et al. (2009), where it is clearly shown that EYFP-tagged ClC-1 is predominantly expressed at T-tubular level in mouse FDB muscle fibers. Third, when considering the question of whether the intracellularly tagged ClC-1 chloride channels in Fig. 9 A colocalize with the RYRs, Lueck et al. (2010) show the green signal from GFP-tagged CLC-1 chloride channels in a “merged” image in Fig. 10 C at a lower intensity than when it is shown in isolation in panel A, whereas the red signal from the RYRs is saturated and is merged at an unchanged intensity level. The reduced intensity of the green signal, together with the fact that the red pixels are saturated, certainly would obscure, rather than reveal, any colocalization of the red and green signals. For proper examination of any colocalization, the green and the red signals need to be of similar intensity to allow one to determine whether the green signals fall between the red pixels or whether they colocalize, in which case the color of the respective pixels would change to yellow (note that only equal amounts of red and green make yellow in additive color mixing). Fourth, we question several aspects related to the electrophysiological measurements in the study by Lueck et al. (2010). For example, the intracellular solution in the voltage clamp experiments did not contain any ATP, even though this has been claimed to greatly influence the properties of ClC-1 (Bennetts et al., 2007; Zhang et al., 2008). There was also no consideration of whether such intracellular conditions affected the mitochondrial and oxidative status of the cell, as the ClC-1 function is known to be affected by oxidative status, and oxidants are known to be produced at the T-system (Hidalgo et al., 2006). The extracellular solution used in the voltage clamp experiments on the adult mouse fibers had an osmolality ∼80 mosmol kg−1 higher than the internal solution (Lueck et al., 2010), and such a disparity was previously shown to increase the rate of charging of the T-system in rat muscle fibers (Lamb, 1986), seemingly by dilating the T-tubules. Both the local generation of oxidants and the dilation of the T-system and consequent membrane stretching could be further reasons why the chloride conductance in the T-system appeared to be small in the present experiments (Lueck et al., 2010). Finally, we note that no mention is made in the article by Lueck et al. (2010), or in the accompanying commentary by Zifarelli and Pusch (2010), about the fact that the data pointing to a tubular location of much of the ClC-1 was obtained in adult rat muscle fibers, which are larger in diameter and have a much more extensive T-tubular system than mouse muscle fibers (Palade and Barchi, 1977; Dulhunty, 1979; Coonan and Lamb, 1998; Pedersen et al., 2004; Dutka et al., 2008). This is of course a critical issue, given that problems with K+ accumulation will be more marked in a larger T-system, as too would be the importance of having Cl− channels localized there rather than solely at the surface. The extensiveness of the T-system in rat muscle fibers, and the difficulty of charging it by current flow from the surface, is shown by its relatively high capacitance and slow charging, which upon imposing a voltage step takes on average ∼6 ms to reach 90% of its final level (e.g., Fig. 7 in Lamb, 1986) compared with less than ∼1.5 ms in adult mouse fibers (Fig. 6 B in Lueck et al., 2010). We also point out that Lueck et al. (2010) offer no explanation for our quite definitive evidence from “skinned” muscle fibers, where the sarcolemma has been removed, that the T-system still retains a large and functionally very important chloride conductance with all the properties of ClC-1; namely, that it is blocked by 9-anthracene carboxylic acid, by low pH, and also by activating PKC with phorbol esters (Coonan and Lamb, 1998; Pedersen et al., 2004; Dutka et al., 2008). (Note that such skinned fiber recordings can be made in

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of β1 integrin, seems to be a key component of FAs, but its exact role in cell–substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin–ILK signaling required for the establishment of cell–substrate adhesion.