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 depolarization-induced but not caffeine-induced Ca ²⁺ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the α _1S subunit is normal, whereas the β _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 β _1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a β _1a -null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the α _1S subunit in the absence of β _1a . Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an ≈2-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 β _1a subunit does not prevent triad targeting of the DHPR α _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 excitation-contraction coupling in β ₁ -null model organisms.

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_V1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca_V1.2 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_V1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca_V1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse–chase labeling showed that membrane-expressed Ca_V1.2-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_V1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca_V1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm²s⁻¹. Consistent with the mobile Ca_V1.2 fraction observed in FRAP, a ∼30% 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_V1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.

Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(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 beta subunits in an isoform-specific manner. Conversely, mutating the alpha-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 beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.

In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca_V1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signal transduction. Here we tested the hypothesis that A-kinase anchoring proteins (AKAPs) or PDZ-proteins are responsible for the targeting and anchoring of Ca_V1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca_V1.2 demonstrated that clusters of membrane-incorporated Ca_V1.2-HA were colocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffold proteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca_V1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca_V1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca_V1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and α-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca_V1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca_V1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. Their number and distribution in dendritic spines is not altered upon NMDA-induced disruption of the glutamate receptor signaling complex, and targeting and anchoring of Ca_V1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

The functional separation between skeletal and cardiac muscles, which occurs at the threshold between vertebrates and invertebrates, involves the evolution of separate contractile and control proteins for the two types of striated muscles, as well as separate mechanisms of contractile activation. The functional link between electrical excitation of the surface membrane and activation of the contractile material (known as excitation–contraction [e–c] coupling) requires the interaction between a voltage sensor in the surface membrane, the dihydropyridine receptor (DHPR), and a calcium release channel in the sarcoplasmic reticulum, the ryanodine receptor (RyR). Skeletal and cardiac muscles have different isoforms of the two proteins and present two structurally and functionally distinct modes of interaction. We use structural clues to trace the evolution of the dichotomy from a single, generic type of e–c coupling to a diversified system involving a novel mechanism for skeletal muscle activation. Our results show that a significant structural transition marks the protochordate to the Craniate evolutionary step, with the appearance of skeletal muscle–specific RyR and DHPR isoforms.

The β subunits of voltage-gated calcium channels regulate surface expression and gating of Ca_V1 and Ca_V2 α₁subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β₄subunit lacking the variable N terminus (β_4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and Ca_V2.1 surface expression in neurons. Compared with the other two known full-length β₄variants (β_4aand β_4b), β_4eis most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β₄subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β₄-null) mice individually reconstituted with β_4a, β_4b, and β_4e. Notably, the number of genes regulated by each β₄splice variant correlated with the rank order of their nuclear-targeting properties (β_4b> β_4a> β_4e). Together, these findings support isoform-specific functions of β₄splice variants in neurons, with β_4bplaying a dual role in channel modulation and gene regulation, whereas the newly detected β_4evariant serves exclusively in calcium-channel-dependent functions.