of bAR on CDI may be limited by basal dephosphorylation processes in TC neurons [9] (see below). Next, we tested agonists which bind preferentially to one of the three bAR subtypes. Therefore xamoterol (b1AR agonist), salmeterol (b2AR agonist), and BRL 37344, (b3AR agonist) were used. Challenging TC neurons with xamoterol (10 mM), reduced the degree of inactivation from 43.862.3% to 37.461.8% (n = 4, p,0.05). This decrease was similar to that induced by salmeterol (10 mM) application (reduction of Dinact from 39.062.2% to 34.560.3%, n = 4, p,0.05; data not shown) and BRL 37344 (10 mM) application (reduction of Dinact from 43.860.6% to 38.360.8%, n = 4, p,0.05; data not shown). To minimize side effects of each drug on non-preferred bAR subtypes and to further enhance the specificity of our pharmacological approach, we used agonists that prefers one type of receptor (in 10 mM concentration) in combination with antagonists for the other two receptor types (in 100 mM concentration), thereby allowing to more selectively investigate the role of each type of receptor in CDI modulation. The following antagonists were applied: CGP 20712 (b1AR antagonist), ICI 118,551 (b2AR antagonist), and SR 59230A (b3AR antagonist). Using this approach, effects on CDI were detected only when the b2AR agonist salmeterol was used in combination with CGP 20712 and SR 59230A (reduction of Dinact from 33.561.2% to 28.461%, n = 4; Figure 4A). For the other two bAR subtypes, no significant modulation of CDI was obtained in this set of experiments (data not shown). These results clearly demonstrate that b2AR specifically contribute to CDI modulation in dLGN TC neurons. Localization of b2ARs in cultured TC neurons Next, we analyzed the specific expression and localization of b2AR in 10 days old cultured thalamic neurons and performed double immunostaining using an antibody against b2AR (Figure 4B) in combination with an antibody against the dendritic marker protein microtubule associated protein 2 (Map2; Figure 4B). The expression of both proteins was detected with b2AR mainly localized in somatic and proximal dendrites of TC neurons (Figure 4B, merge image), cellular compartments that are involved in modulation of CDI in TC neurons. Blocking of PKA suppresses bAR-dependent modulation of CDI in TC neurons Next, we assessed the possible contribution of PKA in CDI modulation of TC neurons by intracellular application of a PKA inhibitor (PKI, 10 mM) in whole cell patch clamp experiments. As shown in Figure 5A, the degree of inactivation was significantly reduced from 44.662.3% (n = 5) under control conditions to 35.161.1% (n = 4, p,0.001; Figure 5A) when TC neurons were challenged with salmeterol (10 mM), while in combination with PKI the inhibitory effect of bAR stimulation was absent (Dinact = 43.261%; n = 5). These findings indicate that the modulation of CDI in TC neurons depends on the activity of PKA. To address the possible association between PKA and CaV1.2, we performed double immunostainings of cultured TC neurons treated with the general bAR agonist isoproterenol. In this set of experiments we used an antibody directed against the regulatory subunit IIb of PKA (PKARIIb), which is highly expressed in thalamus and hippocampus (Figure 5C). Image analysis pointed to a close spatial proximity of the two proteins at the somato- dendritic junction following bAR stimulation (Figure 5B). On the other hand untreated cells showed more somatic expression of the PKARIIb (data not shown). Modulation of Calcium-Dependent Inactivation AKAPs play a crucial role in the modulation of CDI in TC neurons Next, we investigated the possible contribution of AKAP in CDI modulation in TC neurons. AKAP is assumed to simultaneously bind to PKA, Ca2+ channels, and protein phosphatases, including calcineurin [17]. Therefore a selective AKAP inhibitory peptide which binds to the PKA binding sites of AKAP thereby blocking the binding of intracellular PKA and a corresponding control peptide which allows normal PKA binding were used. When salmeterol (10 mM) was applied in the presence of intracellular AKAP inhibitory peptide (10 mM), the degree of inactivation (37.661.5%, n = 9; Figure 6A and 6B) was comparable to control conditions (39.560.5, n = 4, p.0.05) but different from conditions where salmeterol was applied alone (34.560.3%, n = 4, p,0.01; Figure 6A and 6B). Using the same experimental protocol, application of salmeterol in the presence of intracellular control peptide did not change the reduction in CDI (data not shown). In summary, these experiments point to a role of AKAP in the modulation of CDI in TC neurons. Complex formation by the main components of the b-AR signaling cascade in TC neurons After stimulation of the b-AR signaling cascade, it is assumed that a ternary complex is formed between PKA, AKAP, and CaV1.2, the formation of which is important for CDI modulation in TC neurons. In order to find evidence corroborating this assumption, we next performed immunocytochemical staining using antibodies specific for CaV1.2 (Figure 7A, upper left panel), AKAP 5 (Figure 7A, middle left panel), and PKARIIb (Figure 7A, lower left panel), in 10 days old cultured thalamic neurons. Fluorescence imaging revealed that all three proteins are densely expressed in somatic regions and proximal dendrites of TC neurons. Merged images pointed to rather close spatial association of the three proteins (Figure 7A, right panel). In contrary to CaV1.2, another calcium channel CaV2.1 is similarly expressed at somatic regions and proximal dendrites but also additionally in distal dendrites (Figure S2). Protein-protein interactions between components of the ternary inactivation complex Since immunocytochemical stainings pointed to the possibility that PKA, AKAP, and CaV1.2 are located close to each other, thereby contributing to the CDI process, we performed pull down assays which confirmed an interaction between PKARIIb and AKAP7 (Figure 7B) as well as PKARIIb and PKAcsb (Figure 7D). Control incubations of samples of interest with the appropriate fusion-protein partner (empty vector) showed no signal in Western blots. Moreover, indications for an interaction of PKARIIb with another member of the AKAP family, namely AKAP5, were obtained by co-immunoprecipitation experiments. Therefore, magnetic beads coupled to GFP antibody and Western blotting using a PKARIIb-specific antibody were used to verify the binding of PKARIIb to GFP-tagged AKAP5 (Figure 7C). Control incubations of samples of interest (c-myc PKARIIb) with the appropriate fusion-protein partner (empty GFP vector) showed no signal in Western blots. These results provided further evidence for a close coupling between PKA and AKAP in the thalamus. Phosphorylation and dephosphorylation processes in modulation of CDI It is well documented that phosphorylation/dephosphorylation processes play an important role in the regulation of calcium channel activity [3,9]. In an additional series of patch clamp experiments we focused on the role of dephosphorylation in the PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e27474