Subplasmalemmal Ca2+ microdomains formed by brief openings of single or locally clustered Ca2+-permeable ion channels play crucial roles in different cellular processes, including migration and secretion. While electrophysiological recordings are unmatched in sensitivity and temporal resolution, spatiotemporally resolved microfluorometric measurements of such Ca2+ microdomains are of particular interest to identify the sites of channel activity in undisturbed cells under close-to-physiological conditions. In total internal reflection fluorescence (TIRF) microscopy, the shallow penetration depth of the evanescent field is ideally suited to monitor subplasmalemmal Ca2+ microdomains without compromising the spatial and temporal resolution. This study systematically characterizes the impact of various imaging parameters, ion channel properties and cell geometries on the detection limits and signal-to-noise ratios in TIRF microscopy by using simulated and experimentally gathered data. The investigated ion channels include voltage-gated calcium channels, transient receptor potential channels, and P2X receptors. We provide a framework for choosing imaging parameters for high spatiotemporal resolution of Ca2+ influx elicited by openings of single Ca2+-permeable cation channels of different types. Current technical limitations and potential future improvements are discussed.

The K⁺-dependent Na⁺/Ca²⁺-exchanger (NCKX) family comprises five members (NCKX1-5) that serve as key regulators of Ca²⁺ homeostasis. The fourth member, NCKX4, has been implicated in several neuronal processes, including vision, olfaction, and hypothalamic satiety signaling. We recently showed high NCKX4 expression in the mossy fibers of the hippocampus, a brain region essential for learning and memory. In this study, we investigated the impact of NCKX4 loss on Ca²⁺ dynamics in primary hippocampal neurons and assessed hippocampal-dependent behaviour in Nckx4-/- mice. Using Ca2+ imaging, we found that NCKX4 actively contributes to Ca²⁺ homeostasis in hippocampal cells and its loss is not compensated for by other Ca2+ transporters. Neurons lacking NCKX4 had significantly reduced rates of total Na+/Ca2+-exchange activity and showed a decrease in the rate of Ca2+ clearance following a depolarization event. Complementary behavioural testing showed deficits in contextual memory formation and recall, as well as reduced anxiety when placed in an open field, in Nckx4-/- mice. These findings reveal a previously unrecognized role for NCKX4 in regulating hippocampal Ca2+ dynamics, and support the hypothesis that NCKX4 expression is mechanistically linked to learning and memory.