Homozygous zebrafish of the mutant relaxed (red ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarizationinduced but not caffeine-induced Ca 2؉ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the ␣1S subunit is normal, whereas the ␤1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the ␤1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a ␤1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the ␣1S subunit in the absence of ␤1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an Ϸ2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the ␤1a subunit does not prevent triad targeting of the DHPR ␣1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitationcontraction coupling in ␤1-null model organisms.calcium channels ͉ excitation-contraction coupling ͉ tetrads ͉ zebrafish E xcitation-contraction (EC) coupling is understood as the signal transduction process connecting membrane depolarization to the contraction of muscle cells. This process is initiated by the concerted action of two Ca 2ϩ channels, the plasmalemmal voltage-gated dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptor (RyR). In junctions of the SR with the plasma membrane (peripheral couplings) or with the transverse tubules (triads), membrane depolarization is sensed by the DHPR, which then triggers RyR opening and Ca 2ϩ release from the SR. In skeletal muscle cells, this signaltransduction is independent of Ca 2ϩ influx through the DHPR (1) but depends on protein-protein interaction between the DHPR and the RyR1 (2, 3). This physical coupling requires the coordinated arrangement of DHPRs and RyR1s in the junctions. In skeletal muscle triads and peripheral couplings, groups of four DHPRs (tetrads) are arranged in orthogonal arrays matching the opposing RyR1 arrays (4). Formation of DHPR tetrads requires the presence of RyR1.The skeletal muscle DHPR complex is composed of the voltage-sensing and pore-forming ␣ 1S subunit and the auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ (5). Targeted deletions of the ␣ 2 ␦-1 and ␥ subunits do not critically interfere with EC coupling function (6, 7). In contrast, ␣ 1S and ␤ 1a subunit null-mutant mice display a lack of EC coupling and, thus, lethal muscle paralysis (8, 9). Although failure o...

Agonist-triggered downregulation of b-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of b 2 AR signaling highly specific for Ca v 1.2. We find that b 2 -AR binding to Ca v 1.2 residues 1923-1942 is required for b-adrenergic regulation of Ca v 1.2. Despite the prominence of PKA-mediated phosphorylation of Ca v 1.2 S1928 within the newly identified b 2 AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the b 2 AR from Ca v 1.2 upon b-adrenergic stimulation rendering Ca v 1.2 refractory for several minutes from further b-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Ca v 1.2, b 2 AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the b 2 AR from Ca v 1.2 is a uniquely specific desensitization mechanism of Ca v 1.2 regulation by highly localized b 2 AR/cAMP/PKA/ S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Ca v 1.2 and its regulation by channel-associated b 2 AR.

Auxiliary ␤ subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca 2؉ channels in heterologous cells. In brain, all four ␤ isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of ␤ subunits and their role in membrane expression of Ca V 1.2 ␣ 1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous ␤ subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged ␤ subunits demonstrated that all ␤ subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca V 1.2, thus indicating a great promiscuity in ␣ 1 -␤ interactions. In contrast, restricted axonal targeting of ␤ 1 and weak colocalization of ␤ 4b with Ca V 1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca V 1.2 was strongly enhanced by all ␤ subunits in an isoform-specific manner. Conversely, mutating the ␣-interaction domain of Ca V 1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a ␤ subunit with the ␣-interaction domain is absolutely essential for membrane expression of ␣ 1 subunits, as well as for the subcellular localization of ␤ subunits, which by themselves possess little or no targeting properties.Voltage-gated Ca 2ϩ channels (Ca V ) 3 provide key pathways for Ca 2ϩ entry into neurons and translate membrane depolarization into neurotransmitter secretion and gene regulation.Ca V s are composed of a pore-forming ␣ 1 subunit and the auxiliary ␣ 2 ␦ and ␤ subunits (1). Whereas the ␣ 1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in membrane targeting and modulation of channel properties (for review see Ref.2). Presynaptic Ca V s regulate neurotransmitter release (3), and postsynaptic Ca V s activate the transcriptional regulators cAMP-response element-binding protein (CREB) and nuclear factor of activated T-cells (NFAT) (4, 5) and thus modulate long term potentiation (6). These functions reflect both the diversity of Ca V isoforms expressed in brain (7-11) and their differential subcellular localization in neurons (12-15).Four distinct ␤ isoforms have been identified (16 -19), all of which are expressed in brain (20 -23). They contain an Src homology 3 domain and a guanylate kinase domain (24 -27). However, the guanylate kinase fold is modified so that it can bind with high affinity to the so-called ␣-interaction domain (AID) in the intracellular I-II linker of Ca V ␣ 1 subunits (28, 29). The Src homology 3 and the guanylate kinase-like domains are highly conserved among the four genes encoding ␤ subunits (Cacnb1-b4; Fig. 1C), whereas t...

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca V 1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca V 1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca V 1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca V 1.2-SEP) demonstrated ϳ20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca V 1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca V 1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca V 1.2s show highly confined mobility with diffusion coefficients of ϳ0.005 m 2 s Ϫ1 . Consistent with the mobile Ca V 1.2 fraction observed in FRAP, a ϳ30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca V 1.2s maintains stable calcium channel complexes involved in activitydependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.