Modulation of Calcium-Dependent Inactivation Figure 7. Interaction partners important for CDI modulation in TC neurons. (A) Immunocytochemical analysis of primary cultures of the dorsal thalamus using CaV1.2- (red), AKAP150- (green) and PKARIIb- (blue) specific antibodies. Merged picture shows the close connection of the components of the proposed ternary complex, especially in somatic regions and proximal dendrites. Enlarged inlay represents a magnification of the area indicated by the rectangle. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted without signal (negative control). Western blot analysis and pull down assays were done as described in "Methods". (B) Interaction of AKAP7-MBP and PKARIIb-c-myc was detected using antibodies against c-myc. (C) IP of PKARIIb-c-myc and AKAP5-GFP detected after incubation with GFP-coupled magnetic beads using antibodies derived against PKARIIb. (D) Existence of PKA holoenzyme consisting of PKARIIb-GST and PKAcsb-GFP was detected with antibodies against GFP protein. doi:10.1371/journal.pone.0027474.g007 subtypes target PKA to the channel, AKAP5 also targets cal- cineurin and thereby confers unique characteristics upon AKAP5- complexed L-type channels in neurons [17]. AKAP5 is the major AKAP protein in neurons, where it is widely distributed and has been shown to anchor protein kinases and other signaling proteins to multiple receptors and ion channels [48]. On the one side, AKAP5 recruits PKA and calcineurin to the AMPA receptor [49], associates with CaV1.2 [14,50] in neurons, recruits PKA and calcineurin to the channel and is necessary for the bAR sti- mulation of L-type calcium currents [13,14,48] as well as for the L- type calcium current-mediated activation of the transcriptional regulator NFATc4 [17]. On the otherside, AKAP5 binds also to b2AR and facilitates receptor phosphorylation and signaling [51]. Moreover, colocalization with CaV1.2 and postsynaptic density (PSD) proteins in dendritic spines of hippocampal neurons has been shown [52]. Three different binding sites for AKAP5 were described in the N terminus, the cytoplasmic loop connecting repeats I and II, and in the C terminus of CaV1.2 [14]. The C- terminal leucine zipper was shown to be essential for AKAP binding and for bAR stimulation and reversible phosphorylation of CaV1.2 in heart muscle and in neurons [17,47]. Mutation of the three basic residues of this motif blocked AKAP5 and PKA binding and phosphorylation of the Ca2+ channel in response to bAR stimulation [17]. Moreover, mutation of known binding sites Figure 8. The role of dephosphorylation processes in CDI modulation of TC neurons. (A) Representative current traces recorded under control conditions (upper left panel) and in the presence of okadaic acid (OA) (10 mM; left down panel). The bar graph (right panel) represents Dinact under different recording conditions (as indicated). The mean value of three cells recorded under control conditions was taken for comparison with three cells recorded under 10 mM OA. (B) Representative current traces recorded under control conditions (upper left panel) and in the presence of the isoproterenol alone (10 mM; left middle panel) or in combination with OA (10 mM; lower left panel). The bar graph (right panel) represents Dinact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with five cells recorded under 10 mM isoproterenol and five cells recorded under isoproterenol plus OA. Data are presented as means 6 SEM of several independent experiments. ***P,0.001. Significance of isoproterenol plus OA (n = 5) versus controls (n = 5) was calculated by Student’s t test. **P,0.01. Significance of isoproterenol (n = 5) versus isoproterenol plus OA (n = 5) was calculated by Student’s t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. (C) Immunocytochemical analysis of primary cultures of the dorsal thalamus using CaV1.2- (left panel, green) and PP2A-specific antibodies (right panel, red). Merge picture showed close association of the two proteins, especially in somatic regions and proximal dendrites. Data shown are representative pictures from several independent immunostainings and thalamic neurons preparations. In all cases, omission of primary antibodies resulted in no fluorescence signal above background (negative control). doi:10.1371/journal.pone.0027474.g008 PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e27474