Evidence that Ca²⁺ signal transduction by Ca_v1.2 to activate CREB-dependent transcription is mediated by the voltage-gated mobility of the C-tail carrying the CaM-caged Ca²⁺ and is not directly associated with the increase of [Ca²⁺]_i. CREB activation was examined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic channel. (A) Phase-contrast image of the COS1 cell with a shadow of patch pipette. The cell was expressing the -PH_C//α ₂δ-1 channel with the membrane-trapped subunit C-tail, type 1 muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIX domains, both supplemented with nuclear localization sequences. Inset at the bottom: Ca_v1.2- and cAMP-dependent cell signaling pathways mediating CREB-dependent transcription. CBP, CREB-binding protein; CRE, cAMP-response element. (B) Plasma-membrane anchoring of the C-tail (see, schematic diagram) inhibits CREB activation in spite of robust evoked by depolarization. (a) A 100-ms images of FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from = −90 mV. (b) A corresponding representative image of corrected FRET ratio (in a “3D” MetaMorph transformation) recorded within 50-ms windows at −90 and at the end of the last test pulse of the applied train of pulses. (c) An increase of [Ca²⁺]_i detected by Ca²⁺ indicator Fluo4. (d) Representative trace of (20 mM Ca²⁺ in bath medium) evoked by depolarization to +20 mV from = −90 mV showing the sustained component of Ca²⁺ conductance due to the C-tail anchoring. (C) ACh-stimulation of the M1AChR to unbind the C-tail caused activation of IP₃-dependent Ca²⁺ release (panel (c)) but did not show substantial activation of CREB transcription at = −90 mV (panel (a)). (D) CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the C-terminal tail released from the plasma membrane to assume functional voltage-dependent conformation.