L-type Ca2+ (CaV1) channels transduce channel activities into nuclear signals critical to neuritogenesis. Also, standalone peptides encoded by CaV1 DCT (distal carboxyl-terminus) act as nuclear transcription factors reportedly promoting neuritogenesis. Here, by focusing on exemplary CaV1.3 and cortical neurons under basal conditions, we discover that cytosolic DCT peptides downregulate neurite outgrowth by the interactions with CaV1’s apo-calmodulin binding motif. Distinct from nuclear DCT, various cytosolic peptides exert a gradient of inhibitory effects on Ca2+ influx via CaV1 channels and neurite extension and arborization, and also the intermediate events including CREB activation and c-Fos expression. The inhibition efficacies of DCT are quantitatively correlated with its binding affinities. Meanwhile, cytosolic inhibition tends to facilitate neuritogenesis indirectly by favoring Ca2+-sensitive nuclear retention of DCT. In summary, DCT peptides as a class of CaV1 inhibitors specifically regulate the channel activity-neuritogenesis coupling in a variant-, affinity-, and localization-dependent manner.

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. responses to stimuli), and also mediate intracellular signaling cascades that normally take longer time to manifest (e.g., regulations of transcription). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging with long-term probe expression in cortical neurons, which has been designed to eliminate the unwanted interactions between conventional GCaMP indicators and endogenous (apo)calmodulin-binding proteins. By expressing in live adult mice at high levels over an extended time frame, GCaMP-X indicators showed less damage and improved performance in two-photon imaging of acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations. Chronic Ca2+ imaging data (³1 month) were acquired from cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined along with the multiple stages (from neonatal to mature) during neural development. Dysregulations of both neuritogenesis and Ca2+ oscillations were observed typically in 2-3 weeks, which were exacerbated by stronger or prolonged expression of GCaMP. In comparison, neurons expressing GCaMP-X exhibited significantly less damage. By varying the timepoints of virus infection or drug induction, GCaMP-X outperformed GCaMP similarly in cultured mature neurons. These data altogether highlight the unique importance of oscillatory Ca2+ to morphology and health of neurons, presumably underlying the differential performance between GCaMP-X and GCaMP. In summary, GCaMP-X provides a viable option for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

28L-type calcium (CaV1) channels regulate gene expressions via the cascade of excitation-29 transcription coupling, or directly as standalone CCAT (Calcium Channel Associated 30Transcriptional-regulator) peptides encoding distal carboxyl-terminus (DCT) of CaV1, both 31 evidenced in dendritogenesis signaling in neurons. We here discover that DCT peptides opposedly 32 mediate these two sets of transcription signals, all tunable in accordance to C-terminus mediated 33 inhibition (CMI) of Ca 2+ /CaV1 influx. By electrophysiology, neurite morphology, and FRET 2-34 hybrid binding analyses, we systematically examined native and derived DCT peptides across CaV1, 35 unveiling that the overall balance between cytosolic inhibition versus nuclear facilitation is spatially 36 and temporally tuned by CMI of each DCT variant. Our findings not only resolve several 37 controversies existing to DCT variants, but also propose a de novo scheme of CaV1-centric gene 38 regulation: two concurrent routes of transcription signals initiated from either membrane CaV1 39 channels or nuclear CaV1-encoded peptides are subject to autonomous feedback tuning by 40 peptide/channel interactions. 41 42 43 (CDI) (Liu, Yang et al. 2017). It is postulated that CMI should downregulate Ca 2+ /CaV1 signaling 64 along the cascade of excitation-transcription coupling in neurons, in that the conventional inhibitor 65 dihydropyridine (DHP) indeed tunes down CaV1-dependent transcriptional signaling (Redmond, 66 Kashani et al. 2002, Wheeler, Groth et al. 2012). However, if the above hypothesis gets proved, 67 the actual roles of DCT would encounter an apparent dilemma: promotion of neurite outgrowth by 68 regulating related genes as nuclear TF, in direct opposition to inhibition of Ca 2+ influx and 69 presumably also neurite outgrowth as channel inhibitor. In this work, we undertook the mission to 70 elucidate the above controversy regarding CaV1-encoded peptides. 71In neurons, there are two independent sources of peptides encoding DCT fragments. One is 72 from the activation of a cryptic promoter in the coding region of CACNA1C (CaV1.2). Such DCT 73 fragment is termed as calcium channel associated transcriptional regulator (CCATC) and contains 74 intact proximal C-terminal regulatory domain, nuclear retention domain and distal C-terminal 75 regulatory domain (PCRD, NRD and DCRD, respectively) (Figure 2-figure supplement 1) 76 (Gomez-Ospina, Panagiotakos et al. 2013). Here NRD is an indispensable region for Ca 2+ -77 dependent nuclear transport (Gomez-Ospina, Tsuruta et al. 2006), whereas PCRD and DCRD serve 78 as key elements for CMI (Liu, Yang et al. 2017). Two putative transcription activation domains of 79 DCT fragments are distributed on PCRD-NRD junction and DCRD (Gomez-Ospina, Tsuruta et al. 80 2006), the latter of which appears to have more contributions (Gomez-Ospina, Panagiotakos et al. 81 2013). Alternatively, DCT peptides could be generated from proteolytic cleavage of the full-length 82 CaV1. The cleavage sites of amino acid sequences NNAN ...

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