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Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis
The purpose of this study was to quantify the Ca(2+) fluxes underlying Ca(2+) 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 fluoresc...
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2004
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217416/ https://www.ncbi.nlm.nih.gov/pubmed/14676283 http://dx.doi.org/10.1085/jgp.200308908 |
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author | Schuhmeier, R.P. Melzer, W. |
author_facet | Schuhmeier, R.P. Melzer, W. |
author_sort | Schuhmeier, R.P. |
collection | PubMed |
description | The purpose of this study was to quantify the Ca(2+) fluxes underlying Ca(2+) 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). Ca(2+) inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca(2+)-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca(2+) buffer (to represent EGTA), and a sequestration mechanism as Ca(2+) 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 Ca(2+) input fluxes from the measured Ca(2+) transients during depolarizing pulses. In most experiments, intracellular Ca(2+) release dominated the Ca(2+) input flux. In these experiments, the Ca(2+) 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 Ca(2+) 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 Ca(2+) input flux closely resembled the L-type Ca(2+) current, consistent with the absence of SR Ca(2+) release under these conditions and in support of a valid determination of the time course of myoplasmic Ca(2+) input flux based on the optical indicator measurements. |
format | Text |
id | pubmed-2217416 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2004 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22174162008-03-21 Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis Schuhmeier, R.P. Melzer, W. J Gen Physiol Article The purpose of this study was to quantify the Ca(2+) fluxes underlying Ca(2+) 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). Ca(2+) inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca(2+)-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca(2+) buffer (to represent EGTA), and a sequestration mechanism as Ca(2+) 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 Ca(2+) input fluxes from the measured Ca(2+) transients during depolarizing pulses. In most experiments, intracellular Ca(2+) release dominated the Ca(2+) input flux. In these experiments, the Ca(2+) 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 Ca(2+) 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 Ca(2+) input flux closely resembled the L-type Ca(2+) current, consistent with the absence of SR Ca(2+) release under these conditions and in support of a valid determination of the time course of myoplasmic Ca(2+) input flux based on the optical indicator measurements. The Rockefeller University Press 2004-01 /pmc/articles/PMC2217416/ /pubmed/14676283 http://dx.doi.org/10.1085/jgp.200308908 Text en Copyright © 2004, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Schuhmeier, R.P. Melzer, W. Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title | Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title_full | Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title_fullStr | Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title_full_unstemmed | Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title_short | Voltage-dependent Ca(2+) Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis |
title_sort | voltage-dependent ca(2+) fluxes in skeletal myotubes determined using a removal model analysis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217416/ https://www.ncbi.nlm.nih.gov/pubmed/14676283 http://dx.doi.org/10.1085/jgp.200308908 |
work_keys_str_mv | AT schuhmeierrp voltagedependentca2fluxesinskeletalmyotubesdeterminedusingaremovalmodelanalysis AT melzerw voltagedependentca2fluxesinskeletalmyotubesdeterminedusingaremovalmodelanalysis |