Modulation of Calcium-Dependent Inactivation Figure 5. Inhibition of PKA completely suppresses the effect of bAR stimulation on CDI in TC neurons. (A) Representative current traces recorded under control conditions (upper left panel) and in the presence of the specific b2AR agonist salmeterol alone (10 mM; left middle panel) or in combination with PKI 14–22 amide (10 mM; lower left panel). The bar graph represents Dinact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with four cells recorded under 10 mM salmeterol and five cells recorded under 10 mM salmeterol plus PKI 14–22 amide. Data are presented as means 6 SEM of several independent experiments. ***P,0.001. Significance of salmeterol plus PKI (n = 5) versus salmeterol alone (n = 4) 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. (B) Close co-expression of the main modulator of CDI, PKA (green) and CaV1.2 (red) in cultured neurons. Yellow dots represent places were these two proteins are in close proximity. 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). (C) Indicated brain regions were immunostained with antibodies specific for PKARIIb and CaV1.2. Thalamic regions LGN and VB revealed very strong interaction patterns in merged pictures. Association of these proteins is still present in hippocampus but on lower level. DG (dentate gyrus), PoDG (polymorph layer of the dentate gyrus). doi:10.1371/journal.pone.0027474.g005 (Figure 8C, upper left panel). Both proteins were mainly localized in somatic regions and proximal dendrites of TC neurons (Figure 8C, lower panel). Merged pictures pointed to a close spatial arrangement. Discussion The present study provides clear evidence that the CDI of HVA Ca2+channels is decreased by bAR signaling in central neurons. Moreover, we present findings that are in agreement with the existence of a possible protein complex including PKA, AKAP, and CaV1.2 which underlies the modulation of CDI. The most important findings of the present study are: (i) Classical double pulse protocols reveal the occurrence of CDI in TC neurons in a slice preparation; (ii) Activation of b2AR induces the down- regulation of CDI; (iii) Blocking of PKA signaling completely suppresses the effect of bAR stimulation on CDI, (iv) AKAP might play a crucial role in CDI modulation and the docking of PKA to sites close to CaV1.2 (see Text S1). Moreover, blocking of the interaction between AKAP and PKA significantly reduces CDI and translocation of PKA. (v) Phosphorylation and dephosphor- ylation processes represent the basis for bidirectional up- and down-regulation of CDI in TC neurons, respectively. bAR stimulation and modulation of CDI of CaV1.2 via phosphorylation and dephosphorylation processes We have previously demonstrated the existence of CDI of HVA Ca2+ currents in TC neurons in different (acutelyisolated cells, brain slices) thalamic preparations [5,8,9,10]. Based on these findings, the basic features of CDI in TC neurons can be defined as follows: (i) Under control conditions the degree of inactivation varies between about 35–40%. (ii) In addition to L-type Ca2+ channels, Q-type channels are also governed by CDI. (iii) CDI is influenced by a number of cellular mechanisms including repetitive neuronal activity, phosphorylation and dephosphoryla- tion, Ca2+-binding proteins, the cytoskeleton, and intracellular Ca2+ release. The present study adds to these findings by demonstrating the specific influence of b2AR stimulation via PKA and scaffolding proteins of the AKAP family on the degree of CDI. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e27474