Quantitative Analysis of Subcellular Nanodomains Formed by BK and Voltage-Gated Calcium Channels

Abstract

location-dependent, and overlapping, making dissection of this cacophonous phenomenon difficult. Pinpointing the physiological role of solo ion channels is further impeded by a lack in existing technologies capable of determining when and where ion channel types are activated in native tissue. We have generated an endogenous channel activity probe (ECAP) that optically tracks voltage activation of Kv2 type K þ channels. Guided by a conformation-selective, peptide toxin, the fluorescently tagged ECAP binds to resting voltage sensing domain of Kv2 and dissociates from activated voltage sensors. We demonstrate that loss of fluorescent ECAP signal from CA1 neurons in rat hippocampal slices coincides with voltage activation of endogenous Kv2 channels. Confirmatory tests show that the ECAP colocalizes with Kv2-GFP channels in neurons and in cultured cells with or without the presence of the channel's auxiliary subunit AMIGO-1, but not with other channel subtypes. ECAP signal dynamics were both frequency and voltage-stimuli dependent, setting methodical precedence for current and future explorations tracking channel activation or protein conformational changes during physiological signaling.