Analysis of Large Conductance Ca2+‐Activated K+ (BK) Channels Dynamics in Living Cells

Abstract

Large conductance Ca2+‐activated K+ (BK) channels are central in diverse cellular processes including neurotransmitter release or smooth muscle contraction. BK channel activation is driven by both intracellular Ca2+ and voltage, and they are essential for rapid membrane repolarisation. Moreover, BK channels are expressed in human β‐cells regulating insulin secretion. Indeed, in diabetes mellitus BK channels activity is disrupted.Previous electrophysiological studies suggested that N‐type (Cav2.2) calcium‐ and BK‐channel molecules must be in close proximity in the plasma membrane. Therefore, we developed novel methods to analyze the dynamics and distribution of BK channels at the level of single molecules in cell membranes using super‐resolution microscopy, molecular manipulation and electrophysiology. We expressed a cDNA construct that encodes the BK‐channel α‐subunit (BKα) fused to a photoactivatable fluorescent protein mutant of mCherry (PAmCherry) and localized single channel molecules. We also analyzed BK channel localization with gated stimulated emission depletion (g‐STED) microscopy using a BKα‐EGFP construct. Furthermore, we integrated both total internal reflection fluorescence (TIRF) microscopy and patch‐clamp for the study of BK channels function and localization.In order to study Ca2+ channel proximity with BK channels and organelles like secretory vesicles we transfected secretory cells with our BKα‐PAmCherry construct or use the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system to create genetically modified cells, labelling endogenous Cav2.2 channels with fluorescent‐Conotoxin (specific blocker for Cav2.2) that can be photoactivated. With this experiment we could analyse both channel membrane localization and proximity to secretory vesicles and exocytosis sites, using caged Ca2+ to trigger secretory vesicle fusion with the cell membrane. By co‐transfecting cells with both a GCaMP construct localized in the inner cell membrane layer and BKα‐PAmCherry constructs we can track the intracellular Ca2+ rise due to GCaMP fluorescence emission and how depolarization affects BK channels dynamics.These experiments revealed the dynamics, proximity and patterning of large cohorts of single ion channel protein molecules and their functional relationship with the secretory machinery.