Retigabine suppresses loss of force in mouse models of hypokalaemic periodic paralysis.

Recurrent episodes of weakness in periodic paralysis are caused by intermittent loss of muscle fibre excitability, as a consequence of sustained depolarization of the resting potential. Repolarization is favoured by increasing the fibre permeability to potassium. Based on this principle, we tested the efficacy of retigabine, a potassium channel opener, to suppress the loss of force induced by a low-K+ challenge in hypokalaemic periodic paralysis (HypoPP). Retigabine can prevent the episodic loss of force in HypoPP. Knock-in mutant mouse models of HypoPP (Cacna1s p.R528H and Scn4a p.R669H) were used to determine whether pre-treatment with retigabine prevented the loss of force, or post-treatment hastened recovery of force for a low-K+ challenge in an ex vivo contraction assay. Retigabine completely prevents the loss of force induced by a 2 mM K+ challenge (protection) in our mouse models of HypoPP, with 50% inhibitory concentrations of 0.8 ± 0.13 µM and 2.2 ± 0.42 µM for NaV1.4-R669H and CaV1.1-R528H, respectively. In comparison, the effective concentration for the KATP channel opener pinacidil was 10-fold higher. Application of retigabine also reversed the loss of force (rescue) for HypoPP muscle maintained in 2 mM K+. Our findings show that retigabine, a selective agonist of the KV7 family of potassium channels, is effective for the prevention of low-K+ induced attacks of weakness and to enhance recovery from an ongoing loss of force in mouse models of type 1 (Cacna1s) and type 2 (Scn4a) HypoPP. Substantial protection from the loss of force occurred in the low micromolar range, well within the therapeutic window for retigabine.

Voltage-dependent Ca2+ release is impaired in hypokalemic periodic paralysis caused by CaV1.1-R528H but not by NaV1.4-R669H.

Hypokalemic periodic paralysis (HypoPP) is a channelopathy of skeletal muscle caused by missense mutations in the voltage sensor domains (usually at an arginine of the S4 segment) of the CaV1.1 calcium channel or of the NaV1.4 sodium channel. The primary clinical manifestation is recurrent attacks of weakness, resulting from impaired excitability of anomalously depolarized fibers containing leaky mutant channels. Although the ictal loss of fiber excitability is sufficient to explain the acute episodes of weakness, a deleterious change in voltage sensor function for CaV1.1 mutant channels may also compromise excitation-contraction coupling (EC-coupling). We used the low-affinity Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) to assess voltage-dependent Ca2+-release as a measure of EC-coupling for our knock-in mutant mouse models of HypoPP. The peak ΔF/F0 in fibers isolated from CaV1.1-R528H mice was about two-thirds of the amplitude observed in WT mice; whereas in HypoPP fibers from NaV1.4-R669H mice the ΔF/F0 was indistinguishable from WT. No difference in the voltage dependence of ΔF/F0 from WT was observed for fibers from either HypoPP mouse model. Because late-onset permanent muscle weakness is more severe for CaV1.1-associated HypoPP than for NaV1.4, we propose that the reduced Ca2+-release for CaV1.1-R528H mutant channels may increase the susceptibility to fixed myopathic weakness. In contrast, the episodes of transient weakness are similar for CaV1.1- and NaV1.4-associated HypoPP, consistent with the notion that acute attacks of weakness are primarily caused by leaky channels and are not a consequence of reduced Ca2+-release.

Inward rectifier potassium currents in mammalian skeletal muscle fibres.

Inward rectifying potassium (Kir) channels play a central role in maintaining the resting membrane potential of skeletal muscle fibres. Nevertheless their role has been poorly studied in mammalian muscles. Immunohistochemical and transgenic expression were used to assess the molecular identity and subcellular localization of Kir channel isoforms. We found that Kir2.1 and Kir2.2 channels were targeted to both the surface and the transverse tubular system membrane (TTS) compartments and that both isoforms can be overexpressed up to 3-fold 2 weeks after transfection. Inward rectifying currents (IKir) had the canonical features of quasi-instantaneous activation, strong inward rectification, depended on the external [K(+)], and could be blocked by Ba(2+) or Rb(+). In addition, IKir records show notable decays during large 100 ms hyperpolarizing pulses. Most of these properties were recapitulated by model simulations of the electrical properties of the muscle fibre as long as Kir channels were assumed to be present in the TTS. The model also simultaneously predicted the characteristics of membrane potential changes of the TTS, as reported optically by a fluorescent potentiometric dye. The activation of IKir by large hyperpolarizations resulted in significant attenuation of the optical signals with respect to the expectation for equal magnitude depolarizations; blocking IKir with Ba(2+) (or Rb(+)) eliminated this attenuation. The experimental data, including the kinetic properties of IKir and TTS voltage records, and the voltage dependence of peak IKir, while measured at widely dissimilar bulk [K(+)] (96 and 24 mm), were closely predicted by assuming Kir permeability (PKir) values of ∼5.5 × 10(-6 ) cm s(-1) and equal distribution of Kir channels at the surface and TTS membranes. The decay of IKir records and the simultaneous increase in TTS voltage changes were mostly explained by K(+) depletion from the TTS lumen. Most importantly, aside from allowing an accurate estimation of most of the properties of IKir in skeletal muscle fibres, the model demonstrates that a substantial proportion of IKir (>70%) arises from the TTS. Overall, our work emphasizes that measured intrinsic properties (inward rectification and external [K] dependence) and localization of Kir channels in the TTS membranes are ideally suited for re-capturing potassium ions from the TTS lumen during, and immediately after, repetitive stimulation under physiological conditions.

Age-dependent chloride channel expression in skeletal muscle fibres of normal and HSA(LR) myotonic mice.

Abstract We combine electrophysiological and optical techniques to investigate the role that the expression of chloride channels (ClC-1) plays on the age-dependent electrical properties of mammalian muscle fibres. To this end, we comparatively evaluate the magnitude and voltage dependence of chloride currents (ICl), as well as the resting resistance, in fibres isolated from control and human skeletal actin (HSA)(LR) mice (a model of myotonic dystrophy) of various ages. In control mice, the maximal peak chloride current ([peak-ICl]max) increases from -583 ± 126 to -956 ± 260 µA cm(-2) (mean ± SD) between 3 and 6 weeks old. Instead, in 3-week-old HSA(LR) mice, ICl are significantly smaller (-153 ± 33 µA cm(-2)) than in control mice, but after a long period of ∼14 weeks they reach statistically comparable values. Thus, the severe ClC-1 channelopathy in young HSA(LR) animals is slowly reversed with aging. Frequency histograms of the maximal chloride conductance (gCl,max) in fibres of young HSA(LR) animals are narrow and centred in low values; alternatively, those from older animals show broad distributions, centred at larger gCl,max values, compatible with mosaic expressions of ClC-1 channels. In fibres of both animal strains, optical data confirm the age-dependent increase in gCl, and additionally suggest that ClC-1 channels are evenly distributed between the sarcolemma and transverse tubular system membranes. Although gCl is significantly depressed in fibres of young HSA(LR) mice, the resting membrane resistance (Rm) at -90 mV is only slightly larger than in control mice due to upregulation of a Rb-sensitive resting conductance (gK,IR). In adult animals, differences in Rm are negligible between fibres of both strains, and the contributions of gCl and gK,IR are less altered in HSA(LR) animals. We surmise that while hyperexcitability in young HSA(LR) mice can be readily explained on the basis of reduced gCl, myotonia in adult HSA(LR) animals may be explained on the basis of a mosaic expression of ClC-1 channels in different fibres and/or on alterations of other conductances.

Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3.

Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.

Functional expression of transgenic 1sDHPR channels in adult mammalian skeletal muscle fibres.

We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.

Dystrophic skeletal muscle fibers display alterations at the level of calcium microdomains.

The spatiotemporal properties of the Ca(2+)-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca(2+) indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca(2+)-release sites in both mouse strains. The Ca(2+) microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca(2+) microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca(2+)-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca(2+) microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca(2+) transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca(2+)-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca(2+)-release reduction is consistent with that previously measured using global detection techniques.

The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation.

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of CaV1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both CaV1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human CaV1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in CaV1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca2+ release. The prominent role of VSD-I in governing CaV1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished CaV1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of CaV activation, which accounted for both time- and voltage-dependent properties of CaV1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how CaV1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human CaV1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.

Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study.

The double knockout mouse for utrophin and dystrophin (utr(-/-)/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca(2+) transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr(-/-)/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr(-/-)/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr(-/-)/mdx (36%) than in mdx mice (27%), the Ca(2+) release elicited by single stimulation was found to be similarly depressed (32-38%) in utr(-/-)/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr(-/-)/mdx mice, group 1 fibers were able to sustain Ca(2+) release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr(-/-)/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model.

Effects of membrane depolarization and changes in extracellular [K(+)] on the Ca (2+) transients of fast skeletal muscle fibers. Implications for muscle fatigue.

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K(+) gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K(+) K(+)(O) concentration ([K(+)](O)) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca(2+) transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [K(+)](O) and membrane potentials. Depolarization resulting from current injection or raised [K(+](O) produced an increase in the resting [Ca(2+)]. Ca(2+) transients elicited by single stimulation were potentiated by depolarization from -80 to -60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca(2+) transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca(2+) transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca(2+) transients along the trains were associated with impaired or abortive APs. Raising [K(+)](O) to 10 mM potentiated Ca(2+) transients elicited by single and tetanic stimulation, while raising [K(+)](O) to 15 mM markedly depressed both responses. The effects of 10 mM K(+)(O) on Ca(2+) transients, but not those of 15 mM K(+)(O), could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM K(+)(O) might be mediated by depolarization dependent changes in resting [Ca(2+)] and Ca(2+) release, and that additional mechanisms might be involved in the effects of 15 mM K(+)(O) on force generation.

A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers.

Ion movements across biological membranes, driven by electrochemical gradients or active transport mechanisms, control essential cell functions. Membrane ion movements can manifest as electrogenic currents or electroneutral fluxes, and either process can alter the extracellular and/or intracellular concentration of the transported ions. Classic electrophysiological methods allow accurate measurement of membrane ion movements when the transport mechanism produces a net ionic current; however, they cannot directly measure electroneutral fluxes and do not detect any accompanying change in intracellular ion concentrations. Here, we developed a method for simultaneously measuring ion movements and the accompanying dynamic changes in intracellular ion concentrations in intact skeletal muscle fibers under voltage or current clamp in real time. The method combines a two-microelectrode voltage clamp with ion-selective and reference microelectrodes (four-electrode system). We validate the electrical stability of the system and the viability of the preparation for periods of ∼1 h. We demonstrate the power of this method with measurements of intracellular Cl-, H+, and Na+ to show (a) voltage-dependent redistribution of Cl- ions; (b) intracellular pH changes induced by changes in extracellular pCO2; and (c) electroneutral and electrogenic Na+ movements controlled by the Na,K-ATPase. The method is useful for studying a range of transport mechanisms in many cell types, particularly when the transmembrane ion movements are electrically silent and/or when the transport activity measurably changes the intracellular activity of a transported ion.

A highly-selective chloride microelectrode based on a mercuracarborand anion carrier.

The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory glycine and GABAA receptors. Measurement of intracellular chloride activity, [Formula: see text], using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl- ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular [Formula: see text] may be up to 4 times more abundant than Cl- (e.g. skeletal muscle) which places severe constraints on the required selectivity of a Cl- - sensing ISM. Previously, a sensitive and highly-selective Cl- sensor was developed in a polymeric membrane electrode using a trinuclear Hg(II) complex containing carborane-based ligands, [9]-mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of aCl (0.1 mM to 100 mM), is highly selective for Cl- over other biological anions or inhibitors of Cl- transport, and has a 10% to 90% settling  time of 3 sec. Importantly, over the physiological range of aCl (1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to [Formula: see text] or changes in pH. Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular aCl, and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.

Recovery from acidosis is a robust trigger for loss of force in murine hypokalemic periodic paralysis.

Periodic paralysis is an ion channelopathy of skeletal muscle in which recurrent episodes of weakness or paralysis are caused by sustained depolarization of the resting potential and thus reduction of fiber excitability. Episodes are often triggered by environmental stresses, such as changes in extracellular K+, cooling, or exercise. Rest after vigorous exercise is the most common trigger for weakness in periodic paralysis, but the mechanism is unknown. Here, we use knock-in mutant mouse models of hypokalemic periodic paralysis (HypoKPP; NaV1.4-R669H or CaV1.1-R528H) and hyperkalemic periodic paralysis (HyperKPP; NaV1.4-M1592V) to investigate whether the coupling between pH and susceptibility to loss of muscle force is a possible contributor to exercise-induced weakness. In both mouse models, acidosis (pH 6.7 in 25% CO2) is mildly protective, but a return to pH 7.4 (5% CO2) unexpectedly elicits a robust loss of force in HypoKPP but not HyperKPP muscle. Prolonged exposure to low pH (tens of minutes) is required to cause susceptibility to post-acidosis loss of force, and the force decrement can be prevented by maneuvers that impede Cl- entry. Based on these data, we propose a mechanism for post-acidosis loss of force wherein the reduced Cl- conductance in acidosis leads to a slow accumulation of myoplasmic Cl- A rapid recovery of both pH and Cl- conductance, in the context of increased [Cl]in/[Cl]out, favors the anomalously depolarized state of the bistable resting potential in HypoKPP muscle, which reduces fiber excitability. This mechanism is consistent with the delayed onset of exercise-induced weakness that occurs with rest after vigorous activity.

Somatic Ca2+ transients do not contribute to inspiratory drive in preBötzinger Complex neurons.

PreBötzinger Complex (preBötC) neurons are postulated to underlie respiratory rhythm generation. The inspiratory phase of the respiratory cycle in vitro results from preBötC neurons firing synchronous bursts of action potentials (APs) on top of 10-20 mV, 0.3-0.8 s inspiratory drive potentials. Is the inspiratory drive in individual neurons simply the result of the passive integration of inspiratory-modulated synaptic currents or do active processes modulate these currents? As somatic Ca(2+) is known to increase during inspiration, we hypothesized that it affects inspiratory drive. We combined whole cell recording in an in vitro slice preparation with Ca(2+) microfluorometry to detect single inspiratory neuron somatic Ca(2+) transients with high temporal resolution ( approximately mus). In neurons loaded with either Fluo-4 or Oregon Green BAPTA 5 N, we observed Ca(2+) transients associated with each AP. During inspiration, significant somatic Ca(2+) influx was a direct consequence of activation of voltage-gated Ca(2+) channels by APs. However, when we isolated the inspiratory drive potential in active preBötC neurons (by blocking APs with intracellular QX-314 or by hyperpolarization), we did not detect somatic Ca(2+) transients; yet, the parameters of inspiratory drive were the same with or without APs. We conclude that, in the absence of APs, somatic Ca(2+) transients do not shape the somatic inspiratory drive potential. This suggests that in preBötC neurons, substantial and widespread somatic Ca(2+) influx is a consequence of APs during the inspiratory phase and does not contribute substantively to the inspiratory drive potential. Given evidence that the Ca(2+) buffer BAPTA can significantly reduce inspiratory drive, we hypothesize that dendritic Ca(2+) transients amplify inspiratory-modulated synaptic currents.

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers.

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.

Stac3 enhances expression of human CaV1.1 in Xenopus oocytes and reveals gating pore currents in HypoPP mutant channels.

Mutations of CaV1.1, the pore-forming subunit of the L-type Ca2+ channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). However, functional assessment of HypoPP mutant channels has been hampered by difficulties in achieving sufficient plasma membrane expression in cells that are not of muscle origin. In this study, we show that coexpression of Stac3 dramatically increases the expression of human CaV1.1 (plus α2-δ1b and ß1a subunits) at the plasma membrane of Xenopus laevis oocytes. In voltage-clamp studies with the cut-open oocyte clamp, we observe ionic currents on the order of 1 µA and gating charge displacements of ∼0.5-1 nC. Importantly, this high expression level is sufficient to ascertain whether HypoPP mutant channels are leaky because of missense mutations at arginine residues in S4 segments of the voltage sensor domains. We show that R528H and R528G in S4 of domain II both support gating pore currents, but unlike other R/H HypoPP mutations, R528H does not conduct protons. Stac3-enhanced membrane expression of CaV1.1 in oocytes increases the throughput for functional studies of disease-associated mutations and is a new platform for investigating the voltage-dependent properties of CaV1.1 without the complexity of the transverse tubule network in skeletal muscle.

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques.

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F.

BACKGROUND: Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. METHODS: Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. RESULTS: Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd-/-) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. CONCLUSIONS: Alternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865-71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BACKGROUND: Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n',n'-tetraacetic acid (EGTA) to measure Ca(2+) fluxes. Muscles were subjected to both current and voltage clamp conditions. METHODS: Standard and confocal fluorescence microscopy was used to study Ca(2+) release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student's test with significance set at p < 0.05. RESULTS: We found that the peak value of Ca(2+) fluxes elicited by single action potentials was significantly reduced by 15-20 % in C3KO fibers, but the kinetics was unaltered. Ca(2+) release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca(2+) release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca(2+) release in C3KO mice but reduced peak Ca(2+) fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca(2+) release at all triads consistent with a homogenous reduction of functional voltage activated Ca(2+) release sites. CONCLUSIONS: Overall, these results suggest that decreased Ca(2+) release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+.

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K+ concentration in the T-tubules, through its K+ substrate affinity. Apparent K+ affinity was determined from measurements of the K1/2 for K+ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K+ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (-90 to -30 mV) and increased with depolarization in the subthreshold range, -90 to -50 mV. The Q10 was 2.1 over the range of 22-37°C. The K1/2,K of Ip was 4.3±0.3 mM at -90 mV and was relatively voltage independent. This K+ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K+ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K+ increases in proportion to the frequency and duration of action potential firing. This K1/2,K predicts a low fractional occupancy of K+ substrate sites at the resting extracellular K+ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K+ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.