Effects of Axonal Demyelination,Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (I_h) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of I_h (potential of half-maximal activation (V_0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, I_h was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that I_h parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V_0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.     

Reorganization of Thalamic Inputs to Lesioned Cortex Following Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) disrupts thalamic and cortical integrity. The effect of post-injury reorganization and plasticity in thalamocortical pathways on the functional outcome remains unclear. We evaluated whether TBI causes structural changes in the thalamocortical axonal projection terminals in the primary somatosensory cortex (S1) that lead to hyperexcitability. TBI was induced in adult male Sprague Dawley rats with lateral fluid-percussion injury. A virus carrying the fluorescent-tagged opsin channel rhodopsin 2 transgene was injected into the ventroposterior thalamus. We then traced the thalamocortical pathways and analyzed the reorganization of their axonal terminals in S1. Next, we optogenetically stimulated the thalamocortical relays from the ventral posterior lateral and medial nuclei to assess the post-TBI functionality of the pathway. Immunohistochemical analysis revealed that TBI did not alter the spatial distribution or lamina-specific targeting of projection terminals in S1. TBI reduced the axon terminal density in the motor cortex by 44% and in S1 by 30%. A nematic tensor-based analysis revealed that in control rats, the axon terminals in layer V were orientated perpendicular to the pial surface (60.3°). In TBI rats their orientation was more parallel to the pial surface (5.43°, difference between the groups p < 0.05). Moreover, the level of anisotropy of the axon terminals was high in controls (0.063) compared with TBI rats (0.045, p < 0.05). Optical stimulation of the sensory thalamus increased alpha activity in electroencephalography by 312% in controls (p > 0.05) and 237% (p > 0.05) in TBI rats compared with the baseline. However, only TBI rats showed increased beta activity (33%) with harmonics at 5 Hz. Our findings indicate that TBI induces reorganization of thalamocortical axonal terminals in the perilesional cortex, which alters responses to thalamic stimulation.     

Amyloid Prefibrillar Oligomers: The Surprising Commonalities in Their Structure and Activity

It has been proposed that a “common core” of pathologic pathways exists for the large family of amyloid-associated neurodegenerations, including Alzheimer’s, Parkinson’s, type II diabetes and Creutzfeldt–Jacob’s Disease. Aggregates of the involved proteins, independently from their primary sequence, induced neuron membrane permeabilization able to trigger an abnormal Ca²⁺ influx leading to synaptotoxicity, resulting in reduced expression of synaptic proteins and impaired synaptic transmission. Emerging evidence is now focusing on low-molecular-weight prefibrillar oligomers (PFOs), which mimic bacterial pore-forming toxins that form well-ordered oligomeric membrane-spanning pores. At the same time, the neuron membrane composition and its chemical microenvironment seem to play a pivotal role. In fact, the brain of AD patients contains increased fractions of anionic lipids able to favor cationic influx. However, up to now the existence of a specific “common structure” of the toxic aggregate, and a “common mechanism” by which it induces neuronal damage, synaptotoxicity and impaired synaptic transmission, is still an open hypothesis. In this review, we gathered information concerning this hypothesis, focusing on the proteins linked to several amyloid diseases. We noted commonalities in their structure and membrane activity, and their ability to induce Ca²⁺ influx, neurotoxicity, synaptotoxicity and impaired synaptic transmission.     

Co-staining of KCa3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence

The Ca²⁺ activated potassium channel 3.1 (K_Ca3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of K_Ca3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the K_Ca3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of K_Ca3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain K_Ca3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2 . Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained K_Ca3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.     

Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells

Thanks to the crosstalk between Na⁺ and Ca²⁺ channels, Na⁺ and Ca²⁺ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na⁺ (Na_V) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na⁺-Ca²⁺ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the Na_V1.3 channel subtype, which likely endorses their voltage-activated Na⁺ currents. Notably, these Na⁺ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective Na_V1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca²⁺]_i increase. This effect was suppressed by the selective Na_V channel blocker tetrodotoxin, as well by the selective L-type Ca_V channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a Na_V channel blocker, abolished VTD-induced [Ca²⁺]_i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between Na_V and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.     

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca²⁺_i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca²⁺ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca²⁺-induced CaM activation. In the second, the gating occurs without a [Ca²⁺]_i rise.     

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca²⁺]_i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K⁺ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca²⁺-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca²⁺-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca²⁺]_i induced by Chl-T. Both KROS and [Ca²⁺]_i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca²⁺]_i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.     

Role of L-Type Voltage-Gated Calcium Channels in Epileptiform Activity of Neurons

Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 μM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca²⁺]_i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1–10 μM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca²⁺]_i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca²⁺]_i level during oscillations, which appears to determine the number of PDSs in the cluster.     

Finely-Tuned Calcium Oscillations in Osteoclast Differentiation and Bone Resorption

Calcium (Ca²⁺) plays an important role in regulating the differentiation and function of osteoclasts. Calcium oscillations (Ca oscillations) are well-known phenomena in receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption via calcineurin. Many modifiers are involved in the fine-tuning of Ca oscillations in osteoclasts. In addition to macrophage colony-stimulating factors (M-CSF; CSF-1) and RANKL, costimulatory signaling by immunoreceptor tyrosine-based activation motif-harboring adaptors is important for Ca oscillation generation and osteoclast differentiation. DNAX-activating protein of 12 kD is always necessary for osteoclastogenesis. In contrast, Fc receptor gamma (FcRγ) works as a key controller of osteoclastogenesis especially in inflammatory situation. FcRγ has a cofactor in fine-tuning of Ca oscillations. Some calcium channels and transporters are also necessary for Ca oscillations. Transient receptor potential (TRP) channels are well-known environmental sensors, and TRP vanilloid channels play an important role in osteoclastogenesis. Lysosomes, mitochondria, and endoplasmic reticulum (ER) are typical organelles for intracellular Ca²⁺ storage. Ryanodine receptor, inositol trisphosphate receptor, and sarco/endoplasmic reticulum Ca²⁺ ATPase on the ER modulate Ca oscillations. Research on Ca oscillations in osteoclasts has still many problems. Surprisingly, there is no objective definition of Ca oscillations. Causality between Ca oscillations and osteoclast differentiation and/or function remains to be examined.     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

Somatostatin,a Presynaptic Modulator of Glutamatergic Signal in the Central Nervous System

Somatostatin is widely diffused in the central nervous system, where it participates to control the efficiency of synaptic transmission. This peptide mainly colocalizes with GABA, in inhibitory, GABA-containing interneurons from which it is actively released in a Ca²⁺ dependent manner upon application of depolarizing stimuli. Once released in the synaptic cleft, somatostatin acts locally, or it diffuses in the extracellular space through “volume diffusion”, a mechanism(s) of distribution which mainly operates in the cerebrospinal fluid and that assures the progression of neuronal signalling from signal-secreting sender structures towards receptor-expressing targeted neurons located extrasynaptically, in a non-synaptic, inter-neuronal form of communication. Somatostatin controls the efficiency of central glutamate transmission by either modulating presynaptically the glutamate exocytosis or by metamodulating the activity of glutamate receptors colocalized and functionally coupled with somatostatin receptors in selected subpopulations of nerve terminals. Deciphering the role of somatostatin in the mechanisms of “volume diffusion” and in the “receptor-receptor interaction” unveils new perspectives in the central role of this fine tuner of synaptic strength, paving the road to new therapeutic approaches for the cure of central disorders.     

Receptor-Dependent and Independent Regulation of Voltage-Gated Ca2+ Channels and Ca2+-Permeable Channels by Endocannabinoids in the Brain

The activity of specific populations of neurons in different brain areas makes decisions regarding proper synaptic transmission, the ability to make adaptations in response to different external signals, as well as the triggering of specific regulatory pathways to sustain neural function. The endocannabinoid system (ECS) appears to be a very important, highly expressed, and active system of control in the central nervous system (CNS). Functionally, it allows the cells to respond quickly to processes that occur during synaptic transmission, but can also induce long-term changes. The endocannabinoids (eCBs) belong to a large family of bioactive lipid mediators that includes amides, esters, and ethers of long-chain polyunsaturated fatty acids. They are produced “on demand” from the precursors located in the membranes, exhibit a short half-life, and play a key role as retrograde messengers. eCBs act mainly through two receptors, CB1R and CB2R, which belong to the G-protein coupled receptor superfamily (GPCRs), but can also exert their action via multiple non-receptor pathways. The action of eCBs depends on Ca²⁺, but eCBs can also regulate downstream Ca²⁺ signaling. In this short review, we focus on the regulation of neuronal calcium channels by the most effective members of eCBs-2-arachidonoylglycerol (2-AG), anandamide (AEA) and originating from AEA-N-arachidonoylglycine (NAGly), to better understand the contribution of ECS to brain function under physiological conditions.     

GABABR Modulation of Electrical Synapses and Plasticity in the Thalamic Reticular Nucleus

Two distinct types of neuronal activity result in long-term depression (LTD) of electrical synapses, with overlapping biochemical intracellular signaling pathways that link activity to synaptic strength, in electrically coupled neurons of the thalamic reticular nucleus (TRN). Because components of both signaling pathways can also be modulated by GABA_B receptor activity, here we examined the impact of GABA_B receptor activation on the two established inductors of LTD in electrical synapses. Recording from patched pairs of coupled rat neurons in vitro, we show that GABA_B receptor inactivation itself induces a modest depression of electrical synapses and occludes LTD induction by either paired bursting or metabotropic glutamate receptor (mGluR) activation. GABA_B activation also occludes LTD from either paired bursting or mGluR activation. Together, these results indicate that afferent sources of GABA, such as those from the forebrain or substantia nigra to the reticular nucleus, gate the induction of LTD from either neuronal activity or afferent glutamatergic receptor activation. These results add to a growing body of evidence that the regulation of thalamocortical transmission and sensory attention by TRN is modulated and controlled by other brain regions. Significance: We show that electrical synapse plasticity is gated by GABA_B receptors in the thalamic reticular nucleus. This effect is a novel way for afferent GABAergic input from the basal ganglia to modulate thalamocortical relay and is a possible mediator of intra-TRN inhibitory effects.     

Empagliflozin Relaxes Resistance Mesenteric Arteries by Stimulating Multiple Smooth Muscle Cell Voltage-Gated K+ (KV) Channels

The antidiabetic drug empagliflozin is reported to produce a range of cardiovascular effects, including a reduction in systemic blood pressure. However, whether empagliflozin directly modulates the contractility of resistance-size mesenteric arteries remains unclear. Here, we sought to investigate if empagliflozin could relax resistance-size rat mesenteric arteries and the associated underlying molecular mechanisms. We found that acute empagliflozin application produces a concentration-dependent vasodilation in myogenic, depolarized and phenylephrine (PE)-preconstricted mesenteric arteries. Selective inhibition of smooth muscle cell voltage-gated K⁺ channels K_V1.5 and K_V7 abolished empagliflozin-induced vasodilation. In contrast, pharmacological inhibition of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and ATP-sensitive (K_ATP) channels did not abolish vasodilation. Inhibition of the vasodilatory signaling axis involving endothelial nitric oxide (NO), smooth muscle cell soluble guanylyl cyclase (sGC) and protein kinase G (PKG) did not abolish empagliflozin-evoked vasodilation. Inhibition of the endothelium-derived vasodilatory molecule prostacyclin (PGI₂) had no effect on the vasodilation. Consistently, empagliflozin-evoked vasodilation remained unaltered by endothelium denudation. Overall, our data suggest that empagliflozin stimulates smooth muscle cell K_V channels K_V1.5 and K_V7, resulting in vasodilation in resistance-size mesenteric arteries. This study demonstrates for the first time a novel mechanism whereby empagliflozin regulates arterial contractility, resulting in vasodilation. Due to known antihypertensive properties, treatment with empagliflozin may complement conventional antihypertensive therapy.     

The Evidence for Effective Inhibition of INa Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of CaV Channels

Mirogabalin (MGB, Tarlige^®), an inhibitor of the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH₃) cells, we found that MGB was effective at suppressing the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of the voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner, with an effective IC₅₀ of 19.5 and 7.3 μM, respectively, while the K_D value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of I_Na(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of I_Na(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (I_Na(W)) or resurgent I_Na (I_Na(R)) evoked by the respective ascending or descending ramp pulse (V_ramp) was reduced during cell exposure to MGB. MGB-induced attenuation in I_Na(W) or I_Na(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate I_Na. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent I_Na in response to the isosceles triangular V_ramp. The cumulative inhibition of I_Na(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of Ca_V channels, the Na_V channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.     

Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc₂Man₁), while cardiac calsequestrin had five additional mannoses (GlcNAc₂Man₆). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca²⁺-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca²⁺ concentrations, and proper glycosylation, in turn, guarantees both effective Ca²⁺ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.     

Snapin Specifically Up-Regulates Cav1.3 Ca2+ Channel Variant with a Long Carboxyl Terminus

Ca²⁺ entry through Ca_v1.3 Ca²⁺ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Ca_v1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CT_L) of rat Ca_v1.3_L variant. Co-expression of Snapin with Ca_v1.3_L/Ca_vβ₃/α₂δ₂ subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Ca_v1.3_S containing a short CT (CT_S) in which a Snapin interaction site in the CT_L was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Ca_v1.3_L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Ca_v1.3_L. Taken together, our results strongly suggest that Snapin directly interacts with the CT_L of Ca_v1.3_L, leading to up-regulation of Ca_v1.3_L channel activity via facilitating channel opening probability.     

Veratridine Can Bind to a Site at the Mouth of the Channel Pore at Human Cardiac Sodium Channel NaV1.5

The cardiac sodium ion channel (Na_V1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na⁺ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits Na_V1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on Na_V1.5, we perform docking calculations and high-throughput electrophysiology experiments in the present study. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of Na_V1.4 and Na_V1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at Na_V1.5.