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. 2024:2801:189-197.
doi: 10.1007/978-1-0716-3842-2_14.

Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase

Erva Bayraktar  1   2 , Mario Bortolozzi  3   4
Affiliations

Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase

Erva Bayraktar et al. Methods Mol Biol. 2024.

Abstract

The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.

Keywords: Connexin 32; Gating by cytosolic Ca2+; HeLa cells; Hemichannel conductance; Patch-clamp.

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