Whole-cell patch-clamp recording of voltage-sensitive Ca²⁺ channel currents in single cells: heterologous expression...

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 ms. 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.