Local zones of endoplasmic reticulum complexity confine cargo in neuronal dendrites.

Following synthesis, integral membrane proteins dwell in the endoplasmic reticulum (ER) for variable periods that are typically rate limiting for plasma membrane delivery. In neurons, the ER extends for hundreds of microns as an anastomosing network throughout highly branched dendrites. However, little is known about the mobility, spatial scales, or dynamic regulation of cargo in the dendritic ER. Here, we show that membrane proteins, including AMPA-type glutamate receptors, rapidly diffuse within the continuous network of dendritic ER but are confined by increased ER complexity at dendritic branch points and near dendritic spines. The spatial range of receptor mobility is rapidly restricted by type I mGluR signaling through a mechanism involving protein kinase C (PKC) and the ER protein CLIMP63. Moreover, local zones of ER complexity compartmentalize ER export and correspond to sites of new dendritic branches. Thus, local control of ER complexity spatially scales secretory trafficking within elaborate dendritic arbors.

Diversity of dendritic morphology and entorhinal cortex synaptic effectiveness in mouse CA2 pyramidal neurons.

Excitatory synaptic inputs from specific brain regions are often targeted to distinct dendritic arbors on hippocampal pyramidal neurons. Recent work has suggested that CA2 pyramidal neurons respond robustly and preferentially to excitatory input into the stratum lacunosum moleculare (SLM), with a relatively modest response to Schaffer collateral excitatory input into stratum radiatum (SR) in acute mouse hippocampal slices, but the extent to which this difference may be explained by morphology is unknown. In an effort to replicate these findings and to better understand the role of dendritic morphology in shaping responses from proximal and distal synaptic sites, we measured excitatory postsynaptic currents and action potentials in CA2 pyramidal cells in response to SR and SLM stimulation and subsequently analyzed confocal images of the filled cells. We found that, in contrast to previous reports, SR stimulation evoked substantial responses in all recorded CA2 pyramidal cells. Strikingly, however, we found that not all neurons responded to SLM stimulation, and in those neurons that did, responses evoked by SLM and SR were comparable in size and effectiveness in inducing action potentials. In a comprehensive morphometric analysis of CA2 pyramidal cell apical dendrites, we found that the neurons that were unresponsive to SLM stimulation were the same ones that lacked substantial apical dendritic arborization in the SLM. Neurons responsive to both SR and SLM stimulation had roughly equal amounts of dendritic branching in each layer. Remarkably, our study in mouse CA2 generally replicates the work characterizing the diversity of CA2 pyramidal cells in the guinea pig hippocampus. We conclude, then, that like in guinea pig, mouse CA2 pyramidal cells have a diverse apical dendrite morphology that is likely to be reflective of both the amount and source of excitatory input into CA2 from the entorhinal cortex and CA3.

Differential ubiquitination and proteasome regulation of Ca(V)2.2 N-type channel splice isoforms.

Ca(V)2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca(V)2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca(V)2 channels. Here we show that Ca(V)2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca(V)2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca(V)2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a--not e37b--is selected during alternative splicing of Ca(V)2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca(V)2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca(V)2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing.

Team-Based Coaching Intervention to Improve Contrast-Associated Acute Kidney Injury: A Cluster-Randomized Trial.

BACKGROUND: Up to 14% of patients in the United States undergoing cardiac catheterization each year experience AKI. Consistent use of risk minimization preventive strategies may improve outcomes. We hypothesized that team-based coaching in a Virtual Learning Collaborative (Collaborative) would reduce postprocedural AKI compared with Technical Assistance (Assistance), both with and without Automated Surveillance Reporting (Surveillance). METHODS: The IMPROVE AKI trial was a 2×2 factorial cluster-randomized trial across 20 Veterans Affairs medical centers (VAMCs). Participating VAMCs received Assistance, Assistance with Surveillance, Collaborative, or Collaborative with Surveillance for 18 months to implement AKI prevention strategies. The Assistance and Collaborative approaches promoted hydration and limited NPO and contrast dye dosing. We fit logistic regression models for AKI with site-level random effects accounting for the clustering of patients within medical centers with a prespecified interest in exploring differences across the four intervention arms. RESULTS: Among VAMCs' 4517 patients, 510 experienced AKI (235 AKI events among 1314 patients with preexisting CKD). AKI events in each intervention cluster were 110 (13%) in Assistance, 122 (11%) in Assistance with Surveillance, 190 (13%) in Collaborative, and 88 (8%) in Collaborative with Surveillance. Compared with sites receiving Assistance alone, case-mix-adjusted differences in AKI event proportions were -3% (95% confidence interval [CI], -4 to -3) for Assistance with Surveillance, -3% (95% CI, -3 to -2) for Collaborative, and -5% (95% CI, -6 to -5) for Collaborative with Surveillance. The Collaborative with Surveillance intervention cluster had a substantial 46% reduction in AKI compared with Assistance alone (adjusted odds ratio=0.54; 0.40-0.74). CONCLUSIONS: This implementation trial estimates that the combination of Collaborative with Surveillance reduced the odds of AKI by 46% at VAMCs and is suggestive of a reduction among patients with CKD. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: IMPROVE AKI Cluster-Randomized Trial (IMPROVE-AKI), NCT03556293.

Outcomes of cardiac catheterization and percutaneous coronary intervention for in-hospital ventricular tachycardia or fibrillation cardiac arrest.

OBJECTIVE: This study examined outcomes of patients with sudden cardiac death attributable to primary ventricular tachycardia (VT) or ventricular fibrillation (VF) that underwent cardiac catheterization with or without percutaneous coronary intervention (PCI). BACKGROUND: The decision to perform cardiac catheterization and PCI in resuscitated patients with sudden cardiac death remains controversial. Prior data suggest a potential benefit from percutaneous revascularization. METHODS: All patients with an in-hospital pulseless VT or VF cardiac arrest from August 2002 to February 2008 who underwent cardiac catheterization were included. Retrospective chart review was performed to obtain clinical, neurologic, and angiographic data. Primary endpoints were all-cause mortality and neurologic outcome. RESULTS: Two thousand and thirty-four patients had in-hospital cardiac arrest, of these 116 had pulseless VT or VF and were resuscitated and 93 (80%) underwent coronary angiography. The median time to follow-up was 1.3 years (IQR: 0.5-2.9 years). Obstructive coronary artery disease (CAD) was observed in 74 (79%) individuals, of whom 37 underwent PCI. Thirty-five patients with obstructive CAD (47%) died compared to 41% with nonobstructive CAD. In unadjusted and multivariable adjusted analysis PCI was not associated with lower mortality (adjusted hazard ratio: 1.54, 95% CI, 0.79-3.02, P = 0.20). No significant differences were noted in neurologic status at discharge (P = 0.49). CONCLUSION: In this study, an aggressive revascularization strategy with PCI did not confer a survival advantage nor was it associated with improved neurologic outcomes. There was no suggestion of harm with PCI and further studies are necessary to identify potential subgroups that may benefit from revascularization.

Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.

Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.

Pruning and loss of excitatory synapses by the parkin ubiquitin ligase.

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD). The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD.

A Case of Anomalous Left Anterior Descending Artery Originating From the Right Sinus of Valsalva.

The anomalous origin of coronary arteries has been extensively documented in the literature. Most of the anomalies are incidentally found either during coronary angiography or imaging studies and are usually benign; however, malignant outcomes have been reported in the literature. Here, we present the case of a 76-year-old male with non-ST segment elevation myocardial infarction who was found to have an asymptomatic anomalous origin left anterior descending artery from the right sinus of Valsalva.

Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes.

Dendritic spines are micron-sized membrane protrusions receiving most excitatory synaptic inputs in the mammalian brain. Spines form and grow during long-term potentiation (LTP) of synaptic strength. However, the source of membrane for spine formation and enlargement is unknown. Here we report that membrane trafficking from recycling endosomes is required for the growth and maintenance of spines. Using live-cell imaging and serial section electron microscopy, we demonstrate that LTP-inducing stimuli promote the mobilization of recycling endosomes and vesicles into spines. Preventing recycling endosomal transport abolishes LTP-induced spine formation. Using a pH-sensitive recycling cargo, we show that exocytosis from recycling endosomes occurs locally in spines, is triggered by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. Thus, recycling endosomes provide membrane for activity-dependent spine growth and remodeling, defining a novel membrane trafficking mechanism for spine morphological plasticity and providing a mechanistic link between structural and functional plasticity during LTP.

Efficacy of Cangrelor as Bridging Therapy Post PCI.

BACKGROUND: Dual antiplatelet therapy (DAPT) remains the cornerstone management for the prevention of acute stent thrombosis after percutaneous intervention (PCI). Situations mandating early interruption of DAPT carry a high risk of ischemic complications. Perioperative bridge therapy using Cangrelor, an intravenous P2Y2 inhibitor, may offer a potential solution. Unfortunately, evidence for its use in non-cardiac procedures is limited. METHODS: Our protocol demonstrates successful off-label use of IV Cangrelor bridge therapy in a non-cardiac surgery patient. We describe a case of a 77-year old male; triple therapy with Aspirin, Apixaban, and Ticagrelor for recent drug-eluting stent placement required immediate surgical resection of stage I colonic adenocarcinoma. RESULTS: Cangrelor bridge therapy was utilized both preoperatively and postoperatively without ischemic or bleeding complications. The patient tolerated exploratory laparoscopic colectomy with minimal bleeding and good post-op recovery. CONCLUSION: Minimizing the interruption of DAPT therapy in high-risk patients is achievable. However, careful planning with a team-based approach involving surgeons, cardiologists and pharmacists, along with close clinical follow-up and vigilant management of anti-platelet therapy is recommended.

Culprit Vessel Only Versus Multivessel Percutaneous Coronary Intervention in Acute Myocardial Infarction with Cardiogenic Shock: A Systematic Review and Meta-Analysis.

BACKGROUND: Previous studies comparing outcomes between culprit vessel only percutaneous coronary intervention (CV-PCI) versus multivessel percutaneous coronary intervention (MV-PCI) in patients with cardiogenic shock in the setting of acute myocardial infarction have shown conflicting results. This meta-analysis investigates the optimal approach for management of these patients considering recently published data. METHODS: Electronic databases including MEDLINE, ClinicalTrials.gov and the Cochrane Library were searched for all clinical studies published until May 1, 2018, which compared outcomes in patients presenting with acute myocardial infarction and cardiogenic shock. Studies comparing CV-PCI versus MV-PCI in patients with multivessel coronary artery disease were screened for inclusion in final analysis. The primary end point was in-hospital/30 day mortality. Secondary endpoints included long term (>6 months) mortality, renal failure requiring renal replacement therapy, stroke, bleeding, and recurrent myocardial infarction. Odds ratio (OR) with 95% of confidence interval (CI) were computed and p values <0.05 were considered significant. RESULTS: Patient who underwent CV-PCI had significantly lower short-term mortality (in-hospital or 30-day mortality) (OR: 0.73, CI: 0.61-0.87, p = 0.0005), and lower odds of severe renal failure requiring renal replacement therapy (OR: 0.76, CI: 0.59-0.98, p = 0.03). There was no statistically significant difference in long-term mortality, stroke, bleeding, and recurrent myocardial infarction between two groups. CONCLUSION: This meta-analysis showed lower short-term mortality and decreased odds of renal failure requiring renal replacement therapy with CV-PCI compared to MV-PCI. However, subgroup analysis including studies exclusively assessing STEMI patients revealed no statistically significant difference in outcomes. Further randomized trials are needed to confirm these findings and evaluate long term results.

Cell-specific alternative splicing increases calcium channel current density in the pain pathway.

N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.

Phosphatidylinositol-4,5-bisphosphate regulates NMDA receptor activity through alpha-actinin.

Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.

The effect of drug-eluting stents on intermediate angiographic and clinical outcomes in diabetic patients: insights from randomized clinical trials.

OBJECTIVE: Implantation of drug-eluting stents has emerged as the predominant percutaneous revascularization strategy in diabetic patients, despite limited outcomes data. Accordingly, our aim was to conduct a meta-analysis to assess the benefit and safety profile of drug-eluting stents in diabetic patients. METHODS: We included randomized trials comparing either the paclitaxel- or sirolimus-eluting stent with a bare-metal stent or with each other in diabetic patients during a follow-up of at least 6 months. RESULTS: A total of 16 studies were identified, which included 2951 diabetic patients who were followed up for 6 to 12 months. Target lesion revascularization was less frequently performed in patients who received drug-eluting stents compared with bare-metal stents (risk ratio [RR] 0.35, 95% CI 0.27-0.46, P < .0001). Similar reductions were noted in the incidence of major adverse cardiovascular events (RR 0.42, 95% CI 0.31-0.56, P < .0001), in-segment restenosis (RR 0.31, 95% CI 0.25-0.40, P < .0001), and non-Q-wave myocardial infarction (RR 0.57, 95% CI 0.32-0.99, P = .046). Event rates were similar for Q-wave myocardial infarction (RR 0.72, 95% CI 0.25-2.07, P = .54), death (RR 0.64, 95% CI 0.32-1.28, P = .20), and stent thrombosis (RR 0.41, 95% CI 0.13-1.27, P = .12). CONCLUSIONS: In conclusion, diabetic patients who receive drug-eluting stents have a significantly lower incidence of target lesion revascularization, in-segment restenosis and myocardial infarction at 6 to 12 months, compared with bare-metal stents. The rates of mortality and stent thrombosis are similar.

Incremental effect of clopidogrel on important outcomes in patients with cardiovascular disease: a meta-analysis of randomized trials.

OBJECTIVES: To quantify the impact of clopidogrel plus aspirin on the individual outcomes of death, myocardial infarction, or stroke in patients with established cardiovascular disease, or in patients with multiple risk factors for vascular disease. BACKGROUND: Randomized trials have demonstrated a reduction in composite outcomes when clopidogrel is added to aspirin therapy in patients with coronary artery disease; however, the magnitude of benefit on individual outcomes is unknown. METHODS: We conducted a meta-analysis on randomized, controlled trials that compared aspirin plus clopidogrel with aspirin plus placebo for the treatment of coronary artery disease. RESULTS: This analysis included five randomized trials (CURE, CREDO, CLARITY, COMMIT, and CHARISMA) in 79 624 patients. The incidence of all-cause mortality was 6.3% in the aspirin plus clopidogrel group versus 6.7% in the aspirin group (odds ratio [OR] 0.94; 95% CI 0.89, 0.99; p = 0.026). The incidence of myocardial infarction was 2.7% and 3.3% (OR 0.82; 95% CI 0.75, 0.89; p < 0.0001), and stroke was 1.2% and 1.4% (OR 0.82; 95% CI 0.73, 0.93; p = 0.002). Similarly, the incidence of major bleeding was 1.6% and 1.3% (OR 1.26; 95% CI 1.11, 1.41; p < 0.0001), and fatal bleeding was 0.28% and 0.27% (OR 1.04; 95% CI 0.76, 1.43; p = 0.79). CONCLUSION: The addition of clopidogrel to aspirin results in a small reduction in all-cause mortality in patients with ST-elevation myocardial infarction and a modest reduction in myocardial infarction and stroke in patients with cardiovascular disease. The overall incidence of major bleeding however is increased, although there is no excess of fatal bleeds or hemorrhagic strokes.

Valvular Hemolysis Masquerading as Prosthetic Valve Stenosis.

The evaluation of prosthetic valves can provide a unique challenge, and a thoughtful approach is required. High output states like anemia should be kept in the differential when evaluating elevated gradients across prosthetic valves. We present the case of a 69-year-old man with a Starr-Edwards prosthetic aortic valve who presented with symptoms of congestive heart failure and high transvalvular pressure gradients. These symptoms indicate a potential prosthetic valve stenosis. His laboratory evaluation results were consistent with valve-related hemolysis. Resolving his anemia led to a resolution of the symptoms and lowered the pressure gradient on follow-up.

Neuronal L-type calcium channels open quickly and are inhibited slowly.

Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.

What is the risk of stent thrombosis associated with the use of paclitaxel-eluting stents for percutaneous coronary intervention?: a meta-analysis.

OBJECTIVES: This study investigated the risk of stent thrombosis associated with the use of paclitaxel-eluting stents (PES) compared to bare-metal stents (BMS). BACKGROUND: Clinical experience with coronary drug-eluting stents (DES) is relatively limited. There is concern that DES used for percutaneous coronary intervention may result in subsequent thrombosis. METHODS: We conducted a meta-analysis on eight trials (total of 13 study arms) in 3,817 patients with coronary artery disease who were randomized to either PES or BMS. RESULTS: As compared with BMS, PES do not increase the hazard for thrombosis up to 12 months (risk ratio [RR] = 1.06, 95% confidence interval [CI] 0.55 to 2.04, p = 0.86]). There was no evidence of heterogeneity among the studies (chi-square value for Q-statistic = 5.90 [10 degrees of freedom], p = 0.82). Similar results were obtained when the analysis was restricted to trials with a polymeric stent platform (Treatment of de novo coronary disease using a single pAclitaXel elUting Stent [TAXUS]-I, -II, -IV, and -VI) (RR = 1.01, 95% CI 0.40 to 2.53, p = 0.99), trials with longer lesions (TAXUS-IV and -VI) (RR = 0.62, 95% CI 0.2 to 1.91, p = 0.41), and trials that used a higher dose of paclitaxel (ASian Paclitaxel-Eluting Stent Clinical trial [ASPECT], European evaLUaTion of paclitaxel Eluting Stents [ELUTES], and DELIVER-I) (RR = 1.87, 95% CI 0.52 to 6.81, p = 0.34). CONCLUSIONS: Current evidence suggests that standard dose PES do not increase the hazard of stent thrombosis compared to BMS.

Alternative splicing of the beta 4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating.

Ca2+ channel beta subunits are important molecular determinants of the kinetics and voltage dependence of Ca2+ channel gating. Through direct interactions with channel-forming alpha1 subunits, beta subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct beta subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced beta4 subunit containing a short form of domain D1 (beta4a) that is highly homologous to N termini of Xenopus and rat beta3 subunits. The purpose of this study was to compare the gating properties of various alpha1 subunit complexes containing beta4a with those of complexes containing a beta4 subunit with a longer form of domain D1, beta4b. Expression in Xenopus oocytes revealed that, relative to alpha1A and alpha1B complexes containing beta4b, the voltage dependence of activation and inactivation of complexes containing beta4a were shifted to more depolarized potentials. Moreover, alpha1A and alpha1B complexes containing beta4a inactivated at a faster rate. Interestingly, beta4 subunit alternative splicing did not influence the gating properties of alpha1C and alpha1E subunits. Experiments with beta4 deletion mutants revealed that both the N and C termini of the beta4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are alpha1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from beta-subunit splice variant-specific interactions with an alpha1 subunit gating structure.

Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

Ca2+ channel beta subunits modify alpha1 subunit gating properties through direct interactions with intracellular linker domains. In a previous report (Helton and Horne, 2002), we showed that alternative splicing of the beta4 subunit had alpha1 subunit subtype-specific effects on Ca2+ channel activation and fast inactivation. We extend these findings in the present report to include effects on slow inactivation and block by the peptide toxin omega-conotoxin (CTx)-MVIIC. N-terminal deletion and site-directed mutagenesis experiments revealed that the effects of alternative splicing on toxin block and all aspects of gating could be attributed to a proline-rich motif found within N-terminal beta4b amino acids 10-20. Interestingly, this motif is conserved within the third postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain of the distantly related membrane-associated guanylate kinase homolog, PSD-95. Sequence identity of approximately 30% made possible the building of beta4a and beta4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta4b to an extended arm structure that flanks what would be the equivalent of a highly modified PSD-95 carboxylate binding loop. Northern blot analysis revealed a markedly different pattern of distribution for beta4a versus beta4b in the human CNS. Whereas beta4a is distributed throughout evolutionarily older regions of the CNS, beta4b is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta4 subunit has played in the evolution of complex neural networks.