Modulation of Calcium-Dependent Inactivation of L-Type 2+ Ca Channels via b-Adrenergic Signaling in Thalamocortical Relay Neurons Vladan Rankovic1*, Peter Landgraf2, Tatyana Kanyshkova3, Petra Ehling3,4, Sven G. Meuth4, Michael R. Kreutz2, Thomas Budde3., Thomas Munsch1. 1 Institute of Physiology, Otto-von-Guericke University Magdeburg, Germany, 2 PG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany, 3 Institute of Physiology I, Muenster, Germany, 4 Institute of Neuropathophysiology and Neurology Clinic – Department of Inflammatory Disorders of the Nervous System and Neurooncology, Muenster, Germany Abstract Neuronal high-voltage-activated (HVA) Ca2+ channels are rapidly inactivated by a mechanism that is termed Ca2+- dependent inactivation (CDI). In this study we have shown that b-adrenergic receptor (bAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14–22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after bAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via bAR stimulation requires a protein complex consisting of CaV1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca2+ channels allowing their phosphorylation and thereby modulation of CDI. Citation: Rankovic V, Landgraf P, Kanyshkova T, Ehling P, Meuth SG, et al. (2011) Modulation of Calcium-Dependent Inactivation of L-Type Ca2+ Channels via b-Adrenergic Signaling in Thalamocortical Relay Neurons. PLoS ONE 6(12): e27474. doi:10.1371/journal.pone.0027474 Editor: Christoph Kleinschnitz, Julius-Maximilians-Universita¨t W u¨rzburg, Germany Received August 9, 2011; Accepted October 17, 2011; Published December 2, 2011 Copyright: 2011 Rankovic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (GRK 1167-P1 to TM; BU 1019/8-1 to TB; DFG Kr1879/3-1 to MK). VR was a fellow of the GRK 1167, Magdeburg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vladan.rankovic@lin-magdeburg.de . These authors contributed equally to this work. Introduction Voltage-gated Ca2+ channels of the plasma membrane consist three subfamilies (CaV1, CaV2 and CaV3) [1]. They are composed of 10 pore-forming a1 channel subunits and are important components of a universal cellular Ca2+ signaling tool kit [2]. Voltage-dependent Ca2+ channels are one of the main routes of cellular Ca2+ entry. Intracellular Ca2+ ions control processes as diverse as cell proliferation, neuronal development and transmitter release [2]. All of these functions have to be accomplished within a narrow range of Ca2+ concentrations. CDI of voltage-dependent Ca2+ channels is an auto-inhibitory feedback mechanism control- ling Ca2+-influx [3,4]. Previously we have shown that in TC neurons of the dorsal part of the lateral geniculate nucleus (dLGN), Ca2+-induced Ca2+ release (CICR) contributes to intracellular Ca2+ transients [5], leads to the activation of Ca2+-dependent K+ channels and thereby supports regular tonic firing [6]. Further- more, CDI, which is under the control of multiple biochemical and activity-dependent mechanisms, has been shown to limit Ca2+ entry into TC neurons [7,8,9,10]. Stimulation of the bAR/ adenylyl cyclase (AC)/PKA-dependent pathway in TC neurons mediates behavioural state-dependent shifts in thalamic activity modes by modulating pacemaker channels, L-type Ca2+ channels, and Ca2+-dependent K+ channels [3,11]. Direct application of cAMP and the catalytic subunit of PKA reduced the degree of CDI in TC neurons [9]. Although cAMP-dependent signaling and CDI represent prominent mechanisms in TC neuron function, their possible functional coupling by direct stimulation of bAR has not been investigated in these neurons yet. Recent studies in cardiac [12] as well as in hippocampal cells have shown a functional link between bAR and one type of L-type Ca2+ channels, namely CaV1.2, via PKA [13,14], however a possible link to CDI has not been addressed. Furthermore, AKAP has been shown to be an important element in organizing bAR-dependent pathways in neurons [13,15,16]. Although dephosphorylation of Ca2+ channels by calcineurin (PP2B) has originally been proposed to be the crucial mechanism of CDI [17], calmodulin closely tethered to the channel has been identified as the Ca2+ sensor and central mediator of this process [18,19,20]. The role of phosphorylation/dephosphorylation in CDI attracted less attention, although the close association of CaV1.2 channels, phosphorylating PKA, and dephosphorylating calcineurin has been shown [17]. Based on the finding that b2ARs are directly associated with one of their main effector channels, namely CaV1.2, via a protein complex also containing G-proteins, AC, PKA, and a counterbalancing protein phosphatase [13], it was also suggested that this protein complex might be the basis for b-adrenergic modulation of CDI [3] (Figure S1). Here we provide PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e27474