Electrophysiological Approaches for the Study of Ion Channel Function

Abstract

Ion channels play crucial roles in cell physiology, and are a major class of targets for clinically relevant pharmaceuticals. Because they carry ionic current, the function and pharmacology of ion channels can be studied using electrophysiological approaches that range in resolution from the single molecule to many millions of molecules. This chapter describes electrophysiological approaches for the study of one representative ion channel that is defective in a genetic disease, and that is the target of so-called highly effective modulator therapies now used in the clinic: the cystic fibrosis transmembrane conductance regulator (CFTR). Protocols are provided for studying CFTR expressed heterologously, for CFTR expressed in situ in airway epithelial cells, and for purified or partially purified CFTR protein reconstituted into planar lipid bilayers.

Keywords: CFTR; Electrophysiology; Patch clamp; Planar lipid bilayer; Ussing chamber.

Fig. 1

Single channel behavior of CFTR. A representative single-channel current trace is shown for WT-CFTR in the presence of 1 mM MgATP and 127.6 U/mL PKA, from an inside-out membrane patch excised from a Xenopus oocyte, with symmetrical 150 mM Cl⁻. The trace was recorded at V_M = −100 mV. C = closed state; O = full open state. An all-points amplitude histogram is shown under the trace, where the superimposed solid line is results from fit to a Gaussian function

Fig. 2

Macroscopic channel behavior of CFTR from macropatch recording. (Left) A representative macropatch current trace is shown of WT-CFTR recorded in inside-out mode with symmetrical 150 mM Cl⁻ solution. Channels were fully activated in 1 mM MgATP + 127.6 U/mL PKA. A voltage-ramp protocol was applied every 5 s. (Right) Representative macropatch current of WT-CFTR recorded in inside-out mode with a step protocol in the absence (black line) and in the presence (red line) of 50 μM glibenclamide is also shown. Voltage steps from holding potential (0 mV) to +80 mV (a) then −120 mV (b) then +100 mV (c) and back to 0 mV. The blue dashed line indicates zero current level. The time-dependent nature of glibenclamide-mediated channel block (segment b) and relief from block (segments a, c) are evident in comparing the red and black traces

Fig. 3

Macroscopic channel behavior of CFTR from whole-oocyte recording. A representative current trace recorded by two-electrode voltage clamp in ND96 control bath solution is shown, with CFTR activated by exposure to 10 μM Forskolin (FSK). V_M = −60 mV

Fig. 4

Recording of CFTR activity by Ussing chamber technique. Short-circuit currents are measured from 16HBE epithelial cells grown on permeable supports under a chloride gradient. Additions to bathing solution are shown, including Amiloride (20 μM), Albuterol at various concentrations as noted, Forskolin (FSK, 10 μM), and channel inhibitor CFTR_inh172 (10 μM)

Fig. 5

Recording of CFTR reconstituted into a planar lipid bilayer. Shown is a current trace measured at V_M = −60 mV with asymmetric chloride concentration resulting in 100 mV electrochemical driving force. C = closed current level. O = full open state. Currents were filtered at 0.1 kHz