Topic Introduction Patch-Clamp Recording of Voltage-Sensitive Ca 2+ Channels María A. Gandini, 1 Alejandro Sandoval, 2 and Ricardo Felix 1,3 1 Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico; 2 School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla, Mexico In this article, we focus on a refinement of the traditional voltage-clamp methods that are used to measure current from whole cells, or relatively large areas of membrane, called the patch-clamp technique. Although this technique has extended the application of voltage-clamp methods to the recording of ionic currents flowing through single channels, in its whole-cell configuration it has become the most widely used method for recording ionic currents. We give particular attention to the study of voltage-gated (Ca V ) Ca 2+ channels using the patch-clamp technique and discuss some aspects of the molecular physiology of these proteins. INTRODUCTION Much of what we know about the properties of ion channels in cell membranes has come from experiments using the voltage clamp, an experimental method that allows electrophysiologists to hold the voltage of the cell membrane at any preset potential and to measure the currents that flow through the membrane at that potential as a function of time. The first direct recordings of single ion channel currents in biological membranes were made by Neher and Sakmann using an innovative modification of the voltage-clamp method now called the patch-clamp technique (Neher and Sakmann 1976). THE PATCH-CLAMP TECHNIQUE Rather than penetrating the cell with sharp electrodes as is traditionally performed in voltage-clamp experiments, in the patch-clamp technique, blunt-tipped glass pipettes are used in such a way that, when pressed gently against the membrane of a cell, they isolate a small area of membrane. In this way, it is possible to trap or isolate one or a few ion channels in the membrane. The tip of the micropipette is heated to produce a smooth surface that helps in forming a high-resistance seal (>1 GΩ) with the cell membrane (Fig. 1A). Although the interior of the micropipette is filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recordings, or the cytoplasm for whole-cell recordings, the composition of the recording solutions can be changed or drugs can be added to study the ion channels under different experimental conditions. The resistance of the gigaohm seal (or gigaseal) and the use of low-noise devices allows the currents to be electronically isolated and measured across the membrane patch with little competing noise (Hamill et al. 1981). A 3 Correspondence: rfelix@cell.cinvestav.mx © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top066092 329 Cold Spring Harbor Laboratory Press on November 1, 2016 - Published by http://cshprotocols.cshlp.org/ Downloaded from