Numerical Analysis of Ca2+ Depletion in the Transverse Tubular System of Mammalian Muscle

Institute of Physiology and Pathophysiology, Medical Biophysics, University of Heidelberg, INF 326, D-69120 Heidelberg, Germany.
Biophysical Journal (Impact Factor: 3.97). 06/2001; 80(5):2046-55. DOI: 10.1016/S0006-3495(01)76178-4
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Calcium currents were recorded in contracting and actively shortening mammalian muscle fibers. In order to characterize the influence of extracellular calcium concentration changes in the small unstirred lumina of the transverse tubular system (TTS) on the time course of the slow L-type calcium current (I(Ca)), we have combined experimental measurements of I(Ca) with quantitative numerical simulations of Ca2+ depletion. I(Ca) was recorded both in calcium-buffered and unbuffered external solutions using the two-microelectrode voltage clamp technique (2-MVC) on short murine toe muscle fibers. A simulation program based on a distributed TTS model was used to calculate the effect of ion depletion in the TTS. The experimental data obtained in a solution where ion depletion is suppressed by a high amount of a calcium buffering agent were used as input data for the simulation. The simulation output was then compared with experimental data from the same fiber obtained in unbuffered solution. Taking this approach, we could quantitatively show that the calculated Ca2+ depletion in the transverse tubular system of contracting mammalian muscle fibers significantly affects the time-dependent decline of Ca2+ currents. From our findings, we conclude that ion depletion in the tubular system may be one of the major effects for the I(Ca) decline measured in isotonic physiological solution under voltage clamp conditions.

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Available from: Martin Vogel, Mar 26, 2015
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    • "s with time constants ( τ ) of 58 and 150 ms , respectively ( see inset , Fig . 1A ) . The fast tail currents observed at every voltage were significantly larger than the currents observed during the pulses , suggesting that the decay during the pulses did not result from complete inactivation of the Ca 2+ channel , but from other process ( es ) ( Friedrich et al . 2001 ) . Figure 1B shows the voltage dependence of average peak I Ca obtained from control fibres . The resulting I Ca vs . V m plot is asymmetrical , with a negative slope conductance towards a maximal negative peak value ( maximal peak I Ca ) at ∼+35 mV and an ascending limb towards zero current at a E rev of ∼+90 mV . Sizable inward curre"
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    ABSTRACT: Non-technical summary In mammalian skeletal muscle, the coupling between action potential activation and contraction is supposed to be ultimately mediated by the interaction of two ion channels, the L-type calcium channel (so-called dihydropyridine receptor channel) at the transverse tubular system, and the sarcoplasmic reticulum (SR) calcium release channel (so-called ryanodine receptor channel). This paper demonstrates that adult skeletal muscle fibres transfected in vivo with DNA plasmids are able to express functional transgenic dihydropyridine receptor channels. More importantly, the data suggest that transgenic dihydropyridine receptor channels replace native channels in their interaction with SR calcium release channels. Our findings open new avenues for structural and functional studies of the molecular interactions underlying excitation–contraction coupling within the physiologically relevant cellular context of adult mammalian skeletal muscle fibres. Abstract 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, Q and SR Ca2+ 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 them works 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.
    The Journal of Physiology 03/2011; 589(Pt 6):1421-42. DOI:10.1113/jphysiol.2010.202804 · 5.04 Impact Factor
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    • "Indeed, longitudinal tubules also form part of this membrane system. These structures were originally observed by the Italian microscopist E. Veratti at the beginning of the 20th century homeostasis (Almers et al. 1981; Friedrich et al. 2001; Launikonis & Ríos, 2007) and during trafficking of molecules such as insulin and lactate into or out of the cell (Lännergren et al. 1999, 2000; Shorten et al. 2007). The transverse tubules provide the direct link between the extracellular space and the interior of the cell. "
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    ABSTRACT: The tubular (t) system is essential for normal function of skeletal muscle fibre, acting as a conduit for molecules and ions within the cell. However, t system accessibility and interconnectivity have been mainly assessed in fixed cells where the t system no longer fully represents that of the living cell. Here, fluorescent dyes of different diameter were allowed to equilibrate within the t system of intact fibres from toad, mechanically skinned to trap the dyes, and then imaged using confocal microscopy to investigate t system accessibility and interconnectivity. Dual imaging of rhod-2 and a 500 kDa fluorescein dextran identified regions throughout the t system that differed in the accessibility to molecules of different molecular weight. Restrictions within the t system lumen occurred at the junctions of the longitudinal and transverse tubules and also where a transverse tubule split into two tubules to maintain their alignment with Z-lines of adjacent mis-registered sarcomeres. Thus, three types of tubule, transverse, longitudinal and Z, can be identified by their lumenal diameter in this network. The latter we define for the first time as a tubule with a narrow lumen that is responsible for the change in register. Stretch-induced t system vacuolation showed exclusive access of rhod-2 to these structures indicating their origin was the longitudinal tubules. Exposing the sealed t system to highly hypertonic solution reversed vacuolation of longitudinal tubules and also revealed that these tubules are not collapsible. Fluorescence recovery after photobleaching (FRAP) measurements of t system-trapped fluo-5 N showed interconnectivity through the t system along the axis of the fibre. However, diffusion occurred at a rate slower than expected given the known number of longitudinal tubules linking adjacent transverse tubules. This could be explained by the observed narrow opening to the longitudinal tubules from transverse tubules, reducing the effective cross-sectional area in which molecules could move within the t system.
    The Journal of Physiology 10/2008; 586(Pt 21):5077-89. DOI:10.1113/jphysiol.2008.155127 · 5.04 Impact Factor
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    • "As the sealed t system is a closed, finite compartment, any change in [Ca 2+ ] t-sys must be due to Ca 2+ flux across the membrane (Almers et al. 1981; Friedrich et al. 2001) or changes in the volume of the compartment. In the following we neglect the changes in volume (see below; Launikonis & Stephenson, 2004), and assume that J Ca , the net Ca 2+ flux across the t system membrane, is proportional to the rate of change, d[Ca 2+ ] t-sys /dt. "
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    ABSTRACT: Store-operated Ca2+ entry (SOCE) is activated following the depletion of internal Ca2+ stores in virtually all eukaryotic cells. Shifted excitation and emission ratioing of fluorescence (SEER) was used to image mag-indo-1 trapped in the tubular (t) system of mechanically skinned rat skeletal muscle fibres to measure SOCE during intracellular Ca2+ release. Cytosolic Ca2+ transients were simultaneously imaged using the fluorescence of rhod-2. Spatially and temporally resolved images of t system [Ca2+] ([Ca2+]t-sys) allowed estimation of Ca2+ entry flux from the rate of decay of [Ca2+]t-sys. Ca2+ release was induced pharmacologically to activate SOCE without voltage-dependent contributions to Ca2+ flux. Inward Ca2+ flux was monotonically dependent on the [Ca2+] gradient, and strongly dependent on the transmembrane potential. The activation of SOCE was controlled locally. It could occur without full Ca2+ store depletion and in less than a second after initiation of store depletion. These results indicate that the molecular agonists of SOCE must be evenly distributed throughout the junctional membranes and can activate rapidly. Termination of SOCE required a net increase in [Ca2+]SR. Activation and termination of SOCE are also demonstrated, for the first time, during a single event of Ca2+ release. At the physiological [Ca2+]t-sys, near 2 mM (relative to t system volume), SOCE flux relative to accessible cytoplasmic volume was at least 18.6 microM s(-1), consistent with times of SR refilling of 1-2 min measured in intact muscle fibres.
    The Journal of Physiology 09/2007; 583(Pt 1):81-97. DOI:10.1113/jphysiol.2007.135046 · 5.04 Impact Factor
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