Large-conductance calcium- and voltage-activated potassium channels (BK _Ca ) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca ²⁺ ). Under normal cellular conditions, BK _Ca channel activation requires Ca ²⁺ concentrations that typically occur in close proximity to Ca ²⁺ sources. We show that BK _Ca channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BK _Ca -Cav complexes reconstitute a functional “Ca ²⁺ nanodomain” where Ca ²⁺ influx through the Cav channel activates BK _Ca in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BK _Ca -mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild‐type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno‐associated viruses (AAVs), each containing half of the otoferlin cDNA. Co‐injecting these dual‐AAV2/6 half‐vectors into the cochleae of 6‐ to 7‐day‐old otoferlin knock‐out ( Otof ^−/− ) mice led to the expression of full‐length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual‐AAV transduction of Otof ^−/− IHCs fully restored fast exocytosis, while otoferlin‐dependent vesicle replenishment reached 35–50% of wild‐type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40–60 dB. Therefore, we propose that gene delivery mediated by dual‐AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF .

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.