The delayed rectifier potassium conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers.

A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the transverse tubular system (TTS) were used to characterize delayed rectifier K currents (IK(V)) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IK(V) displays the canonical hallmarks of K(V) channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gK(V)) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IK(V). Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IK(V), but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IK(V) records. A two-channel model that faithfully simulates IK(V) records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ∼31% of gK(V), and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IK(V)1.4 and IK(V)3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IK(V) resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IK(V) records. Normalized peak attenuations showed the same voltage dependence as peak IK(V) plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IK(V) and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gK(V) in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that K(V) channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IK(V) arises from the TTS

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

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 β1a 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

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 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 DiBAC4(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 DiBAC4(5). The peak ΔF/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 DiBAC4(5) exhibit ΔF/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

Calcium Release Domains in Mammalian Skeletal Muscle Studied with Two-photon Imaging and Spot Detection Techniques

The spatiotemporal characteristics of the Ca2+ 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 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] 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 (∼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 Ca2+ transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest ΔF/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 Ca2+ 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 ∼1.4 ms and dissipate within ∼4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca2+ transients peaked at T-tubule locations, was 0.62 μm, similar to the 0.61 μm 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 Ca2+ domains in adult mammalian muscle fibers are confined to Ca2+ release sites located at the junctional sarcoplasmic reticulum (SR)

Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers

The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally

The Na conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers

Na (and Li) currents and fluorescence transients were recorded simultaneously under voltage-clamp conditions from mouse flexor digitorum brevis fibers stained with the potentiometric dye di-8-ANEPPS to investigate the distribution of Na channels between the surface and transverse tubular system (TTS) membranes. In fibers rendered electrically passive, voltage pulses resulted in step-like fluorescence changes that were used to calibrate the dye response. The effects of Na channel activation on the TTS voltage were investigated using Li, instead of Na, because di-8-ANEPPS transients show anomalies in the presence of the latter. Na and Li inward currents (INa, ILi; using half of the physiological ion concentration) showed very steep voltage dependences, with no reversal for depolarizations beyond the calculated equilibrium potential, suggesting that most of the current originates from a noncontrolled membrane compartment. Maximum peak ILi was ∼30% smaller than for INa, suggesting a Li-blocking effect. ILi activation resulted in the appearance of overshoots in otherwise step-like di-8-ANEPPS transients. Overshoots had comparable durations and voltage dependence as those of ILi. Simultaneously measured maximal overshoot and peak ILi were 54 ± 5% and 773 ± 53 µA/cm2, respectively. Radial cable model simulations predicted the properties of ILi and di-8-ANEPPS transients when TTS access resistances of 10–20 Ωcm2, and TTS-to-surface Na permeability density ratios in the range of 40:60 to 70:30, were used. Formamide-based osmotic shock resulted in incomplete detubulation. However, results from a subpopulation of treated fibers (low capacitance) provide confirmatory evidence that a significant proportion of ILi, and the overshoot in the optical signals, arises from the TTS in normal fibers. The quantitative evaluation of the distribution of Na channels between the sarcolemma and the TTS membranes, as provided here, is crucial for the understanding of the radial and longitudinal propagation of the action potential, which ultimately govern the mechanical activation of muscle in normal and diseased conditions

Membranes for Solar Water Splitting Devices

Abstract not Available

Potassium-sensitive loss of muscle force in the setting of reduced inward rectifier K+ current: Implications for Andersen–Tawil syndrome

Andersen-Tawil syndrome (ATS) is an ion channelopathy with variable penetrance for the triad of periodic paralysis, arrhythmia, and dysmorphia. Dominant-negative mutations of KCNJ2 encoding the Kir2.1 potassium channel subunit are found in 60% of ATS families. As with most channelopathies, episodic attacks in ATS are frequently triggered by environmental stresses: exercise for periodic paralysis or stress with adrenergic stimulation for arrhythmia. Fluctuations in K+, either low or high, are potent triggers for attacks of weakness in other variants of periodic paralysis (hypokalemic periodic paralysis or hyperkalemic periodic paralysis). For ATS, the [K+] dependence is less clear; with reports describing weakness in high-K+ or low-K+. Patient trials with controlled K+ challenges are not possible, due to arrhythmias. We have developed two mouse models (genetic and pharmacologic) with reduced Kir currents, to address the question of K+-sensitive loss of force. These animal models and computational simulations both show K+-dependent weakness occurs only when Kir current is <30% of wildtype. As the Kir deficit becomes more severe, the phenotype shifts from high-K+-induced weakness to a combination where either high-K+ or low-K+ triggers weakness. A K+ channel agonist, retigabine, protects muscle from K+-sensitive weakness in our mouse models of the skeletal muscle involvement in ATS