Patch-Clamp Recordings from Native Cells and Isolation of Membrane Currents

The development of the “gigaseal” patch-clamp technique revolutionized the application of whole-cell current- and voltage-clamp recording methods to the analyses of native cells. As in experiments on heterologously expressed channels, after the formation of a gigaseal, the patch of membrane under the recording pipette is disrupted, while keeping the pipette–cell seal intact, providing direct, low-resistance access to the cell interior and enabling membrane potential (current clamp) or current (voltage clamp) recordings to be obtained. The whole-cell recording technique provides control of the intracellular, as well as the extracellular, solutions, facilitating the isolation of membrane currents. This chapter discusses and illustrates the application of whole-cell voltage-clamp recording methods to identify/characterize the various ionic currents coexpressed in native cells, using (mouse) cardiac myocytes as the exemplar. In addition to demonstrating the isolation of inward and outward currents, select voltage-clamp paradigms and pharmacological tools are applied to illustrate approaches to identify kinetically distinct voltage-gated K+ (Kv) current components coexpressed in the same cell. Experimental approaches to identify the molecular determinants of native mouse myocardial Kv currents are also presented. In addition, the potential of combining whole-cell recordings with dynamic-clamp and action potential-clamp methods to explore the roles of individual Kv currents in shaping action potentials and, conversely, the impact of variations in action potential trajectories in shaping the waveforms of evoked currents in native cells, are explored.