Voltage-dependent Ca2+ Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis

Abstract

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the “removal model fit” approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 μM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at −16 and −20 mV, respectively, compared with −2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 μM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

“…The Ca 2ϩ occupancies of all model compartments [T-sites, P-sites, ATP (F-sites) and uptake] were summed, and the release flux was calculated as the time derivative of the sum. Calculations were performed using Euler's method (36). Analysis software was written in Delphi (Borland) and Visual Basic for Excel (Microsoft).…”

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle

“…The other parameter values were set as follows: [Dye] total , [EGTA] total and [BAPTA] total were set proportionally to the concentrations in the pipette and an exponential ‘entry’ function of time, namely where a is a small negative constant of convenience and τ equals 37.5 min for BAPTA and 34.8 min for EGTA. These values were adapted from an average estimate of EGTA entry by Schuhmeier & Melzer (2004). The slight difference between time constants of BAPTA and EGTA was calculated assuming inverse proportionality between τ and diffusion coefficient D and inverse proportionality between D and molecular radius, which was calculated from the molecular weight.…”

“…The kinetic constants of EGTA:Ca were k on = 15 (μ m s) −1 and k off = 7.5 s −1 (Schuhmeier & Melzer, 2004; Royer et al 2008). For BAPTA:Ca they were k on = 1000 (μ m s) −1 and k off = 200 s −1 (Wu et al 1996).…”

A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca 2+ release in muscle fibers of Y522S RyR1 knock-in mice

“…To construct Ca 2ϩ current-voltage relationships, we averaged the last 10 milliseconds of each depolarization step. Ratiometric Ca 2ϩ transients were subjected to a removal model fit analysis as originally described by Melzer et al (28) to calculate the flux of Ca 2ϩ from the SR (11,29). The pipette concentrations of EGTA and fura-2 were used as the buffer concentrations for the removal analysis.…”