Whole-cell patch-clamp recording of voltage-sensitive Ca2+ channel currents in single cells: Heterologous expression systems and neurones

Jon Brown, Atticus H. Hainsworth, Alessandro Stefani, Andrew D. Randall

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Voltage-sensitive calcium channels (VSCC) are vital to the normal physiology of many cell types, including neurones, skeletal, cardiac and smooth muscle cells, heart pacemaker tissue and endocrine cells. Whole-cell recording is a functional electrophysiological assay that allows real-time measurement of macroscopic VSCC activity at the level of single cells. Using this technique, it is possible to probe the molecular physiology, pharmacology, and biophysics of VSCC proteins with a level of precision rarely surpassed in cell biological studies. With best practice voltage-dependent gating behaviors of VSCCs can be interrogated with temporal resolution >100 μs. These advantages have commonly been exploited using recombinant channels heterologously expressed in cell-lines, where the molecular identity of the channel under study can be precisely defined, and also in native cell types freshly isolated from intact tissue using enzymes. The latter approach is especially valuable for study of adult brain neurons as these cells are not amenable to primary culture. We also describe a method with which VSCCs can be studied in nucleated macropatches derived from neurons without the use of enzymes. Although automated patch-clamp systems are now available they have limitations, and manual whole-cell recording of VSCC currents remains an expert technique requiring intelligent, conative experimentation.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages123-148
Number of pages26
Volume937
ISBN (Print)9781627030854
DOIs
Publication statusPublished - 2013

Publication series

NameMethods in Molecular Biology
Volume937
ISSN (Print)10643745

Keywords

  • Calcium channel
  • Calcium channel alpha1 subunit
  • Calcium channel antagonist
  • Calcium current
  • CaV
  • HVA
  • LVA
  • Whole-cell patch clamp

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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