Abstract
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.
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Acknowledgments
Work in the laboratory of D.A.J. has been supported by National Institutes of Health Grants K01DK081666, R01DK097392, Vanderbilt DRTC Pilot and Feasibility Grant P60DK20593, ADA grant 1-17-IBS-024 (D.A.J.).; Vanderbilt METP grant 5T32DK07563, National Institutes of Health Grant 1F31DK109625 (N.C.V.); Vanderbilt ITED grant T32DK101003 (M.T.D.). Previous work in the Philipson lab was also supported by R01DK092616-01A1 (P.I.M.W. Roe) and P30DK020595 (G.I. Bell) and R01DK48494 (L.H. Philipson).
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Vierra, N.C., Dickerson, M.T., Philipson, L.H., Jacobson, D.A. (2018). Simultaneous Real-Time Measurement of the β-Cell Membrane Potential and Ca2+ Influx to Assess the Role of Potassium Channels on β-Cell Function. In: Shyng, SL., Valiyaveetil, F., Whorton, M. (eds) Potassium Channels. Methods in Molecular Biology, vol 1684. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7362-0_7
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DOI: https://doi.org/10.1007/978-1-4939-7362-0_7
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