Simultaneous Real-Time Measurement of the β-Cell Membrane Potential and Ca2+ Influx to Assess the Role of Potassium Channels on β-Cell Function.

Stimulus-secretion coupling in pancreatic β-cells requires Ca2+ influx through voltage-dependent Ca2+ channels, whose activity is controlled by the plasma membrane potential (V m). Here, we present a method of measuring fluctuations in the β-cell V m and Ca2+ influx simultaneously, which provides valuable information about the ionic signaling mechanisms that underlie insulin secretion. This chapter describes the use of perforated patch clamp electrophysiology on cells loaded with a fluorescent intracellular Ca2+ indicator, which permits the stable recording conditions needed to monitor the V m and Ca2+ influx in β-cells. Moreover, this chapter describes the protocols necessary for the preparation of mouse and human islet cells for the simultaneous recording of V m and Ca2+ as well as determining the specific islet cell type assessed in each experiment.