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

Stimulus-secretion coupling in pancreatic β-cells requires Ca influx through voltage-dependent Ca channels, whose activity is controlled by the plasma membrane potential (V ). Here, we present a method of measuring fluctuations in the β-cell V and Ca 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 Ca indicator, which permits the stable recording conditions needed to monitor the V and Ca 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 and Ca as well as determining the specific islet cell type assessed in each experiment.