Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Small Molecules as Modulators of Voltage-Gated Calcium Channels in Neurological Disorders: State of the Art and Perspectives.

Voltage-gated calcium channels (VGCCs) are widely expressed in the brain, heart and vessels, smooth and skeletal muscle, as well as in endocrine cells. VGCCs mediate gene transcription, synaptic and neuronal structural plasticity, muscle contraction, the release of hormones and neurotransmitters, and membrane excitability. Therefore, it is not surprising that VGCC dysfunction results in severe pathologies, such as cardiovascular conditions, neurological and psychiatric disorders, altered glycemic levels, and abnormal smooth muscle tone. The latest research findings and clinical evidence increasingly show the critical role played by VGCCs in autism spectrum disorders, Parkinson's disease, drug addiction, pain, and epilepsy. These findings outline the importance of developing selective calcium channel inhibitors and modulators to treat such prevailing conditions of the central nervous system. Several small molecules inhibiting calcium channels are currently used in clinical practice to successfully treat pain and cardiovascular conditions. However, the limited palette of molecules available and the emerging extent of VGCC pathophysiology require the development of additional drugs targeting these channels. Here, we provide an overview of the role of calcium channels in neurological disorders and discuss possible strategies to generate novel therapeutics.

Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development.

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on α2δ1 knockdown. Overexpression of α2δ3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons.

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Phosphorylation of Cav1.2 on S1928 uncouples the L-type Ca2+ channel from the ß2 adrenergic receptor.

Agonist-triggered downregulation of ß-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of ß2AR signaling highly specific for Cav1.2. We find that ß2-AR binding to Cav1.2 residues 1923-1942 is required for ß-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified ß2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the ß2AR from Cav1.2 upon ß-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further ß-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, ß2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the ß2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized ß2AR/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 Cav1.2 and its regulation by channel-associated ß2AR.

Differential neuronal targeting of a new and two known calcium channel ß4 subunit splice variants correlates with their regulation of gene expression.

The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß 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 ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) 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 CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.

Stable incorporation versus dynamic exchange of ß subunits in a native Ca2+ channel complex.

Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary ß subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether ß subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and ß subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle ß1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle ß2a and ß4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of ß relative to the α1 subunit, altered the specific dynamic properties of the ß subunits. In contrast, a single residue substitution in the α interaction pocket of ß1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous ß1a subunit forms a stable complex, whereas the heterologous ß2a and ß4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the ß subunit isoforms.

Surface traffic of dendritic CaV1.2 calcium channels in hippocampal neurons.

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 ∼20% 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 ∼0.005 µm² s⁻¹. Consistent with the mobile Ca(V)1.2 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(V)1.2s 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.

Reciprocal interactions regulate targeting of calcium channel beta subunits and membrane expression of alpha1 subunits in cultured hippocampal neurons.

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.

Resolving sub-synaptic compartments with double immunofluorescence labeling in hippocampal neurons.

Immunofluorescence microscopy of synaptic proteins is a powerful and commonly used approach in cellular neurosciences. Many studies use green/red color overlays of immunofluorescence images to demonstrate synaptic co-localization of an unknown protein with a known synaptic marker. However, this approach fails to identify the specific sub-synaptic compartment in which a protein is localized. Here we describe a novel analysis method to determine the precise location of proteins within synapses of cultured hippocampal neurons with double immunofluorescence staining. This approach is based on center-to-center distance measurements of fluorescent clusters of protein pairs coexisting in synapses. We validated the method by analyzing the distances between different combinations of well-established synaptic marker proteins. The results demonstrate that protein pairs in the active zone and the postsynaptic density, two sub-synaptic compartments which are separated by less than 50 nm, can be readily distinguished from each other and from marker pairs co-localized within a single sub-synaptic compartment. Thus, center-to-center distance analysis can resolve the distance across the synaptic cleft and it is useful for localizing synaptic proteins to specific pre- and postsynaptic compartments.

Stable membrane expression of postsynaptic CaV1.2 calcium channel clusters is independent of interactions with AKAP79/150 and PDZ proteins.

In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca(V)1.2 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(V)1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca(V)1.2 demonstrated that clusters of membrane-incorporated Ca(V)1.2-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(V)1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca(V)1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca(V)1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and alpha-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca(V)1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca(V)1.2 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(V)1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

Evolution of skeletal type e-c coupling: a novel means of controlling calcium delivery.

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 beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.

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 alpha(1S) subunit is normal, whereas the beta(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 beta(1a) protein in mutant muscle. Thus, the immotile zebrafish relaxed is a beta(1a)-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the alpha(1S) subunit in the absence of beta(1a). Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an approximately 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 beta(1a) subunit does not prevent triad targeting of the DHPR alpha(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 beta(1)-null model organisms.