Measuring sarcoplasmic reticulum Ca2+ content, fractional release, and Ca2+ buffering in cardiac myocytes

Niall Macquaide, Virginie Bito, Karin R. Sipido*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Here, we describe a protocol for the reliable measurement of the amount of Ca(2+) in the sarcoplasmic reticulum (SR) Ca(2+) store of cardiac myocytes. The whole-cell patch-clamp method is used to provide controlled loading of the SR during conditioning depolarizing pulses, followed by rapid application of a high dose of caffeine to release all SR Ca(2+) and to prevent Ca(2+) reuptake by the SR. Simultaneous measurement of membrane currents records Ca(2+) extruded through the Na(+)-Ca(2+) exchanger. The integral of the caffeine-induced Na(+)-Ca(2+) exchange current is then used as a measure of the SR Ca(2+). Derived measurements include the Ca(2+) buffering capacity and measurement of fractional release as an indicator of coupling gain. Caveats, advantages, and disadvantages of this method and alternative methods are discussed.

Original languageEnglish
Pages (from-to)403-7
Number of pages5
JournalCold Spring Harbor protocols
Volume2015
Issue number4
DOIs
Publication statusPublished - 1 Apr 2015

Keywords

  • Animals
  • Buffers
  • Calcium/metabolism
  • Myocytes, Cardiac/metabolism
  • Patch-Clamp Techniques/methods
  • Sarcoplasmic Reticulum/metabolism

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