Functional Microstructure of CaV-Mediated Calcium Signaling in the Axon Initial Segment.

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (CaV). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of CaVs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: CaV3 generates hotspot regions of calcium influx coupled to calcium stores, whereas CaV2 channels underlie diffuse calcium influx between hotspots. Therefore, different CaV classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.

Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow.

Determining the mechanism of action (MOA) of novel or naturally occurring compounds mostly relies on assays tailored for individual target proteins. Here we explore an alternative approach based on pattern matching response profiles obtained using cultured neuronal networks. Conolidine and cannabidiol are plant-derivatives with known antinociceptive activity but unknown MOA. Application of conolidine/cannabidiol to cultured neuronal networks altered network firing in a highly reproducible manner and created similar impact on network properties suggesting engagement with a common biological target. We used principal component analysis (PCA) and multi-dimensional scaling (MDS) to compare network activity profiles of conolidine/cannabidiol to a series of well-studied compounds with known MOA. Network activity profiles evoked by conolidine and cannabidiol closely matched that of ω-conotoxin CVIE, a potent and selective Cav2.2 calcium channel blocker with proposed antinociceptive action suggesting that they too would block this channel. To verify this, Cav2.2 channels were heterologously expressed, recorded with whole-cell patch clamp and conolidine/cannabidiol was applied. Remarkably, conolidine and cannabidiol both inhibited Cav2.2, providing a glimpse into the MOA that could underlie their antinociceptive action. These data highlight the utility of cultured neuronal network-based workflows to efficiently identify MOA of drugs in a highly scalable assay.

A high-throughput assay for evaluating state dependence and subtype selectivity of Cav2 calcium channel inhibitors.

Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.