Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRa), and (iii) histone binding through association of Cacnb4 with HP1c concomitantly with Ser 10 histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 μM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.

Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

Maurocalcine (MCa) is a 33-amino acid peptide isolated from the venom of Scorpio maurus palmatus, a Tunisian scorpion. It possesses efficient cell penetration properties and has been shown to strongly modify ryanodine receptor (RyR1) channel behavior by increasing open probability and inducing a long-lasting subconductance state. The amino acid residue threonine at position 26 belongs to a putative phosphorylation site within MCa sequence. We investigated the effect of a) T26 phosphorylation (MCa T26Phospho) by protein kinase A and b) replacement of T26 by glutamic acid residue to mimic phosphorylation on the effect of MCa on RyR1 properties. Using [ 3 H]ryanodine ([ 3 H]Ry) binding and single channel voltage-clamp measurements, we show that both MCa T26Phospho and MCa T26E analogs almost completely lose the ability to induce RyR1 activation. Neither MCa T26Phospho nor MCa T26E (up to 2mM) increase [ 3 H]rya binding. Small increase (~1.65-fold activation; p<0.01) was observed with high concentrations (10mM) of either MCa T26Phospho or MCa T26E. Single channel measurements revealed that neither MCa T26E nor MCa T26Phospho (up to 2mM) were able to induce the characteristic long-lasting subconductance state of RyR1 observed in presence of wild-type MCa. MCa T26Phospho altered RyR1 channel gating in a timedependent biphasic manner with initial activation of Po followed by almost complete inactivation. These effects of MCa T26Phospho could be reversed by 200nM wt MCa. At concentrations R2mM, MCa T26E enhances Po~3 fold by shortening t c without altering t o. These results show that single phosphorylation of MCa modifies RyR1 activity.1P01 AR52354.

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