Modulation of Calcium-Dependent Inactivation Figure 6. Inhibition of PKA binding to AKAPs suppresses the effect of bAR stimulation 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 InCELLectTM AKAP St-Ht31 inhibitory peptide (10 mM; lower left panel). (B) The bar graph represents Dinact under different recording conditions (as indicated). The mean value of four cells recorded under control conditions was taken for comparison with four cells recorded under 10 mM salmeterol and nine cells recorded under 10 mM salmeterol plus InCELLectTM AKAP St-Ht31 inhibitory peptide. Data are presented as means 6 SEM of several independent experiments. **P,0.01. Significance of salmeterol (n = 4) versus salmeterol plus InCELLectTM AKAP St-Ht31 inhibitory peptide (n = 9) 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. doi:10.1371/journal.pone.0027474.g006 In the present study, we demonstrated that stimulation of bAR, which leads to phosphorylation of Ca2+ channels in a cAMP- dependent manner via PKA activation, significantly reduced the degree of inactivation of L-type Ca2+ channels. The same results were obtained when channel dephosphorylation was inhibited. This indicates that phosphorylation keeps L-type Ca2+ channels in a state of high open probability, ready to open during depolarization [9,25]. The modulation of L-type Ca2+ channels through phosphorylation via different second messenger systems is well established, and includes phosphorylation by PKA and CaM kinase II (for review, see [26,27]). Both types of modulation result in an increase in peak current amplitude [28,29]. Furthermore, CDI has been shown to be reduced by activation of cAMP- dependent phosphorylation [9,25,30]. Therefore, both the incre- ment in HVA Ca2+ current amplitude and the reduction of CDI after bAR stimulation observed in the present study are consistent with a phosphorylation of L-type Ca2+ channels by PKA. The serine residue at position 1928 (Ser1928) of CaV1.2 channels is one important target of PKA activity in heart and brain [31,32,33]. However, it has been recently shown that mutation of Ser1928 of cardiac L-type Ca2+ channels has only a small effect on channel phosphorylation after bAR stimulation [34], pointing to the existence of additional phosphorylation sites [34,35,36]. Moreover, meaningful regulation of channel activity by PKA phosphorylation requires a proper balance with depho- sphorylation processes. Several studies in hippocampal neurons have shown that protein phosphatases including PP1, PP2A, and calcineurin directly bind to the C-terminal region of CaV1.2 [17,31,33]. Moreover, the signaling pathway from b2AR to the CaV1.2 channel, including G-proteins, adenylate cyclase, PKA and the counterbalancing protein phosphatases, forms a closely associated protein complex in the forebrain [13]. However, the role of this complex in context of CDI modulation has not been addressed yet. CaV1.2 channels are expressed in TC neurons [8] and the existence of bARs, coupled positively to adenylate cyclase in these neurons, has been shown [37]. Moreover, we previously demonstrated effects of calyculin A and ascomycin, which are blockers of PP1, PP2A, and calcineurin [38] on CDI and Ca2+ current amplitude in TC neurons [9]. The present study on brain slices confirmed and extended previous findings obtained in acutely isolated TC neurons by identifying b2AR as the receptor subtype involved in CDI modulation and showing that blocking protein phosphatases by okadaic acid, has a significant effect on CDI during b-AR stimulation. From our data, it is therefore reasonable to conclude that PKA and protein phosphatases antagonistically modulate CDI of CaV1.2 channels in TC neurons via phosphorylation and dephosphorylation processes. Although our experiments with okadaic acid demonstrated the role of dephosphorylation processes in modulation of CDI after bAR stimulation in TC neurons, the specific type of phosphatase involved in TC neurons is still not clear. The original model of CDI in Helix aspersa included a dephosphorylation cycle by calcineurin as the fundamental step leading to channel closure [39]. Later, evidence for and against an involvement of calcineurin was found [17,40,41,42,43,44,45]. In TC neurons, application of the calcineurin blocker ascomycin boosts CDI [9,46]. Although these observations clearly indicate a modulation of CDI by calcineurin in TC neurons, the inactivation process itself is probably not a dephosphorylation reaction. As shown above, PKA is the main enzyme which phosphorylates CaV1.2 channels, thereby keeping them in an open state (increasing their open probability) and restraining the effects of CDI. Besides this phosphorylation processes that occur after bAR stimulation, there might be a constant dephosphorylation driven by PP1 and PP2. AKAP mediates the modulation of CaV1.2 channel during bAR stimulation CaV1.2 channels can physically associate with either AKAP5 or AKAP7 through a leucine zipper interaction [17,47]. Both, the modulation of channels and its downstream signaling depend upon the identity of the associated AKAPs. Although both AKAP PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e27474