Downloaded from http://cshprotocols.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Topic Introduction Patch-Clamp Recording of Voltage-Sensitive Ca2+ Channels María A. Gandini,1 Alejandro Sandoval,2 and Ricardo Felix1,3 1Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico; 2School 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 2+ the study of voltage-gated (CaV)Ca 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 3Correspondence: [email protected] © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top066092 329 Downloaded from http://cshprotocols.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press M.A. Gandini et al. A Feedback resistor Amplifier Voltage output Electrode Current measurement Voltage command Ground Ground Cell Voltage-gated calcium channel Glass micropipette Recording External Cell Ca2+ chamber solution membrane Internal solution High-resistance Cell electrical seal B Pull Inside-out Cell-attached excised patch Suction Pull Outside-out Whole-cell excised patch FIGURE 1. Experimental setup for conducting the patch-clamp technique. (A) Sample cell immersed in external recording solution within a recording chamber attached via a glass micropipette that forms a gigaseal with the plasma membrane. The micropipette contains an internal recording solution connecting the cell interior to the feedback resistor and recording apparatus via a silver electrode. Current flow measured during a patch-clamp experiment is equal and opposite to the micropipette current. (B) Different configurations of the conventional patch-clamp technique allowing recordings to be made on excised patches: the inside-out mode (top-right), made by pulling the membrane patch off the cell into the bath solution, and the outside-out mode (lower-right), made by applying suction (lower-left) to destroy the membrane isolated by the patch pipette and then pulling the pipette away from the cell. silver wire coated with silver chloride is placed in contact with the solution in the micropipette and conducts electric current to the amplifier. Several variants of the basic patch-clamp technique can be applied according to the needs of the research plan (Hamill et al. 1981). Many studies have used patches in the cell-attached mode, but the resting potential of the cell is not known and neither the intracellular nor the extracellular ion concentrations can be changed easily. For these reasons, it is sometimes essential to work using a cell-free mode, with patches excised from the main body of the cell (Fig. 1B). There are two kinds of 330 Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top066092 Downloaded from http://cshprotocols.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Patch Clamping of CaV Channels recordings on excised patches: (i) the inside-out mode, made by pulling the membrane patch off the cell into the bath solution and (ii) the outside-out mode, made by applying suction to destroy the membrane isolated by the patch pipette and then pulling the pipette away from the cell. In this case, the membrane should reseal to give a patch of membrane whose intracellular face is in contact with the micropipette solution. Finally, the whole-cell recording is achieved by destroying the membrane patch using suction so that the cell, whose interior then comes into contact with the solution in the micropipette, can be voltage- or current-clamped (Marty and Neher 1995). The cell contents equilibrate over time with the solution within the micropipette. In the perforated patch variant of the whole-cell recording, the experimenter forms a gigaseal but does not destroy the patch membrane. Instead, the electrode solution contains an antifungal or antibiotic agent (nystatin or gramicidin) that diffuses and forms small perforations in the membrane patch, providing electrical access to the cell interior (Fig. 1B). The whole-cell patch clamp has now largely replaced high-resistance microelectrode recording techniques to record currents across the entire cell membrane. The main electronic components of a traditional patch-clamp setup include an operational am- plifier, a personal computer, an analog-to-digital converter interface, a micropipette manipulator, a microscope, either upright or inverted, in most cases equipped for phase-contrast and fluorescence, a perfusion system, a vibration isolation table, and a Faraday cage. The single most important electronic component of a patch-clamp amplifier is the current-to-voltage converter, which is contained in the headstage and has a high gain, owing to the large feedback resistor. It can be arranged so that the potential inside the micropipette can either be held at a steady level or changed in a stepwise fashion. In principle, current flow through the electrode across a resistor of high impedance causes a voltage drop that is proportional to the measured micropipette current. An operational amplifier is used to automatically adjust the voltage source to maintain a constant micropipette potential at the desired reference potential. As the response of the amplifier is fast, it can be assumed that, for all practical purposes, the micropipette potential is proportional to the reference potential. When current flows across the membrane through ion channels, the micropipette potential is instantaneously displaced from the reference potential. The amplifier alters the voltage source to generate a micro- pipette current that will exactly oppose the displacement of the micropipette potential from the reference potential. Thus, the current flow measured during a patch-clamp experiment is equal and opposite to the micropipette current. As discussed below, Ca2+ currents in many cells can be characterized in patch-clamp experiments by the mode of activation and the unitary conductance of the channels, as well as by selective blockade by drugs, ions, or toxins. FUNCTIONAL DIVERSITY OF CaV CHANNELS CaV channels are a family of transmembrane proteins widely distributed in excitable cells and also found at low levels in many nonexcitable cells. These channels open when the plasma membrane becomes depolarized and mediate Ca2+ entry in response to action potentials and subthreshold 2+ depolarizing signals. Ca entering the cell through CaV channels serves as the second messenger of electrical signaling, initiating a variety of cellular events, including neurotransmitter release, muscle contraction and gene expression, among many others (Catterall 2011). Soon after the first recordings of Ca2+ currents, it was apparent that there were multiple types of Ca2+ currents which have been defined subsequently by physiological and pharmacological criteria. In cardiac, smooth, and skeletal muscle, the major Ca2+ currents are distinguished by high voltage of activation, large single-channel conductance, slow voltage-dependent inactivation, and specific inhi- bition by antagonist drugs including dihydropyridines (Tsien et al. 1988). As these currents show slow voltage-dependent inactivation and