Full-length Channel Research Articles

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Effect of heat-up and oxidation on the PHWR channel under severe accident scenario-an experimental study

During unmitigated postulated severe accidents, the coolant channels of the pressurized heavy water reactor are heated to elevated temperatures. The channels may further get exposed to oxidizing media during the accident progression. Both heat-up rate and oxidation reaction affect the sagging deformation of channels. This study explores the effect of initial transient heat-up rate on the channel deformation and the contact time between heated and un-heated channels experimentally. The effect of oxidizing environment on the structural integrity of the channels is also studied. As experimentation with multiple full-length channels with bundle weights is complex and expensive, the experiments are conducted with scaled-down channels with geometric scaling of 1:3. During experiments, the sagging deformation of the channel has increased gradually with a low heat-up rate, whereas the contact between the channels has been instantaneous at a high heat-up rate. It is thus suggested that the deformation of the heated channel and the contact propagation along contacting channels may be governed by the rate-dependent plasticity (creep). The oxidative reaction in oxygen-rich corrosive media has severely deteriorated the outer calandria tube of the coolant channel, exposing the inner pressure tube to corrosion. The oxidation of the pressure tube degrades its mechanical strength resulting in the failure of the whole coolant channel. The length of central debris supported by the unheated bottom channel is long (equivalent to 3–4 fuel bundles) compared to end debris. The insight generated from this experimental study will further be used to validate the computational models for the channel deformation and its associated structural integrity.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

Pannexin1 (PANX1) is a large-pore ATP efflux channel with a broad distribution, which allows the exchange of molecules and ions smaller than 1 kDa between the cytoplasm and extracellular space. In this study, we show that in human macrophages PANX1 expression is upregulated by diverse stimuli that promote pyroptosis, which is reminiscent of the previously reported lipopolysaccharide-induced upregulation of PANX1 during inflammasome activation. To further elucidate the function of PANX1, we propose the full-length human Pannexin1 (hPANX1) model through cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulation studies, establishing hPANX1 as a homo-heptamer and revealing that both the N-termini and C-termini protrude deeply into the channel pore funnel. MD simulations also elucidate key energetic features governing the channel that lay a foundation to understand the channel gating mechanism. Structural analyses, functional characterizations, and computational studies support the current hPANX1-MD model, suggesting the potential role of hPANX1 in pyroptosis during immune responses.

Mutational effects of Pannexin 1 R217H depend on the carboxyl-terminus

Pannexin 1 (Panx1) has been implicated in a plethora of physiological and pathophysiological processes. It is one of the major ATP release channels in many cell types. Extracellular ATP, activates purinergic P2X and P2Y receptors, triggering several signaling cascades. A disease-associated mutation, Arg-217-His (R217H) in the 3rd transmembrane domain of Panx1 attenuates channel functions through an unknown mechanism. Since carboxyl terminus (CT) gates the channel, we hypothesized that R217 interacts with the CT, and this interaction is required for optimum channel activities. R217H mutation though reduced the currents in the full-length channel, did not affect CT-truncated Panx1-Δ386. Also, compared to the wild-type, Panx1-R217H expressing cells showed lesser cell death when activated through P2X7 receptor. However, cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells were similar. The mutation is ineffective unless the channel has an intact CT. Based on our results we propose that R217H mutation perturbs the conformational flexibility of CT, leading to channel dysfunction.

A Homology Modelling Study of Potential RyR2 Structural Changes in R2474S and V2475F CPVT1

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a disease caused by single-point mutations to cardiac ryanodine receptor Ca2+ release channels (RyR2). Life-threatening arrhythmias due to abnormal sarcoplasmic reticulum Ca2+ release can result. of the many CPVT1-causing mutations, two distinct CPVT1 gain-of-function mutations are found at neighbouring amino acid residues, R2474S and V2475F in the Helical Domain 1 (HD1), more than 43 Å from the transmembrane pore. To explore potential changes in the RyR2 HD1 structure caused by these mutations, we used MODELLER to construct HD1 homology models for the human wild-type (WT) as well as the two mutants, R2474S and V2475F. The models were based on resolved structures of the pig RyR2 HD1 (residues 2111 to 2679) from full-length channels in open and closed states (PDB ID: 5GOA, 5GO9). Subsequently, we generated a root mean square deviation (RMSD) matrix of modelled structures and used hierarchical clustering on the distance matrix to assess differences caused by mutants. Our results indicate that both mutations may alter the bending of the HD1 domain, particularly in the open state. From the full-length structures, it has previously been suggested that the motions of the HD1 are coupled to motions of the central domain (including the pore region). Thus, shifts in the HD1 may be translated into changes in the pore. As both mutations are reported to increase RyR2 activity, we suggest that these potential structural changes within the RyR2 HD1 domain are sufficient to subsequently alter the interactions between this domain and other regions of the channel so that channel opening is encouraged.

DESIGN AND INVESTIGATION OF WEIGHT BUNDLE SIMULATOR FOR INDIAN PHWR USING APDL - A THERMAL ASPECT

Under a postulated scenario of Loss of Coolant Accident (LOCA) with un-availability of emergency core cooling system (ECCS) for Indian Pressurized Heavy Water Reactor (IPHWR), the channel integrity needs to be assured. An experimental facility is presently being developed at Indian Institute of Technology Roorkee (IIT R) India to study such a severe event. In this study a CFD simulation of fuel bundle weight simulator is being carried out using ANSYS 19.0 in transient thermal analysis using ANSYS Parametric Design Language (APDL) solver. The test facility aims to estimate the thermal aspect of weight bundle simulator which is the source of heat generation for pressure tube. Thermo-mechanical deformation of pressure tube will depend on the heat source, therefore it is of great importance to check the thermal integrity of weight bundle fuel simulator. Power requirement, weight, and dimensions of weight bundle fuel simulator is similar to the actual fuel bundle used in 700 MWe IPHWR. Simulation was done for 3 % and 2% decay heat. From analysis it was found that the rate of temperature rise in pressure tube with 3 % decay heat reached a maximum of 2 0C/sec and average rate of temperature rise was around 1 0C/sec whereas with 2% decay heat, pressure tube maximum temperature rise was 1.5 0C/sec and average rate 0.8 0C/sec. Results obtained will be further used for designing of 700 MWe full length channel set-up.

Calmodulin binds to Drosophila TRP with an unexpected mode

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Intracellular activation of full-length human TREK-1 channel by hypoxia, high lactate, and low pH denotes polymodal integration by ischemic factors.

TREK-1, a two-pore domain potassium channel, responds to ischemic levels of intracellular lactate and acidic pH to provide neuroprotection. There are two splice variants of hTREK1: the shorter splice variant having a shorter N-terminus compared with the full-length hTREK1 with similar C-terminus sequence that is widely expressed in the brain. The shorter variant was reported to be irresponsive to hypoxia-a condition attributed to ischemia, which has put the neuroprotective role of hTREK-1 channel into question. Since interaction between N- and C-terminus of different ion channels shapes their gating, we re-examined the sensitivity of the full-length as well as the shorter hTREK-1 channel to intracellular hypoxia along with lactate. Single-channel data obtained from the excised inside-out patches of the full-length channel expressed in HEK293 cells indicated an increase in activity as opposed to a decrease in activity in the shorter isoform. However, both the isoforms showed an increase in activity under combined hypoxia, 20mM lactate, and low pH6 condition, albeit with subtle differences in their individual actions, confirming the neuroprotective role played by hTREK-1 irrespective of the differences in the N-terminus among the splice variants. Furthermore, E321A mutant that disrupts the interaction of the C-terminus with the membrane showed a decrease in activity with hypoxia indicating the importance of the C-terminus in the hypoxic response of the full-length hTREK-1. We propose an increase in activity of both the splice variants of hTREK-1 in combined hypoxia, high lactate, and low pH conditions typically associated with ischemia provides neuroprotection.

Electrophysiological characterization of the modified hERGT potassium channel used to obtain the first cryo-EM hERG structure.

The voltage‐gated hERG (human‐Ether‐à‐go‐go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss‐of‐function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain‐of‐function mutations lead to short QT syndrome. A recent open channel cryo‐EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure‐function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full‐length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole‐cell measurements of hERG current (IhERG) were made at 37°C from HEK 293 cells expressing wild‐type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end‐pulse nor tail IhERG density significantly differed between WT and hERGT. However, the IhERG deactivation rate was significantly slower for hERGT. Half‐maximal activation voltage (V0.5) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT, but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady‐state “window” current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain.

CaV1.3 L-Type Calcium Channels Increase the Vulnerability of Substantia Nigra Dopaminergic Neurons in MPTP Mouse Model of Parkinson's Disease.

Mechanisms underlying the selective vulnerability of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) over those in the ventral tegmental area (VTA) to degeneration in Parkinson’s disease (PD) remain poorly understood. DA neurons of SNpc and VTA are autonomous pacemakers but pacemaking in SNpc but not in VTA is accompanied by calcium influx through L-type calcium channel, CaV1.3 contributing to increased intracellular calcium and hence to cell death. CaV1.342A, an alternatively spliced short variant of CaV1.3 has increased calcium influx. We, therefore studied the role of CaV1.342 (full-length channel) and CaV1.342A in mouse SNpc in PD pathogenesis by quantifying mRNA levels of CaV1.342 and CaV1.342A in SNpc and followed the change in their levels in MPTP induced parkinsonism mouse model. Using in situ hybridization and immunohistochemistry we observed the localization of mRNA of CaV1.342 and CaV1.342A in tyrosine hydroxylase (TH) positive DA neurons. Further, mRNA levels of CaV1.342A were higher in SNpc as compared to the cortex. Upon MPTP treatment, mRNA levels of CaV1.342 and CaV1.342A maintained their levels in SNpc in spite of the loss of ~50% of the DA neurons. This indicates that the expression of CaV1.342 and CaV1.342A is maintained at a robust level during the degenerative process in the parkinsonism model.

Valproic acid interactions with the NavMs voltage-gated sodium channel

Valproic acid (VPA) is an anticonvulsant drug that is also used to treat migraines and bipolar disorder. Its proposed biological targets include human voltage-gated sodium channels, among other membrane proteins. We used the prokaryotic NavMs sodium channel, which has been shown to be a good exemplar for drug binding to human sodium channels, to examine the structural and functional interactions of VPA. Thermal melt synchrotron radiation circular dichroism spectroscopic binding studies of the full-length NavMs channel (which includes both pore and voltage sensor domains), and a pore-only construct, undertaken in the presence and absence of VPA, indicated that the drug binds to and destabilizes the channel, but not the pore-only construct. This is in contrast to other antiepileptic compounds that have previously been shown to bind in the central hydrophobic core of the pore region of the channel, and that tend to increase the thermal stability of both pore-only constructs and full-length channels. Molecular docking studies also indicated that the VPA binding site is associated with the voltage sensor, rather than the hydrophobic cavity of the pore domain. Electrophysiological studies show that VPA influences the block and inactivation rates of the NavMs channel, although with lower efficacy than classical channel-blocking compounds. It thus appears that, while VPA is capable of binding to these voltage-gated sodium channels, it has a very different mode and site of action than other anticonvulsant compounds.

A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane.

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

Elucidating a role for the cytoplasmic domain in the Mycobacterium tuberculosis mechanosensitive channel of large conductance

Microbial survival in dynamic environments requires the ability to successfully respond to abrupt changes in osmolarity. The mechanosensitive channel of large conductance (MscL) is a ubiquitous channel that facilitates the survival of bacteria and archaea under severe osmotic downshock conditions by relieving excess turgor pressure in response to increased membrane tension. A prominent structural feature of MscL, the cytoplasmic C-terminal domain, has been suggested to influence channel assembly and function. In this report, we describe the X-ray crystal structure and electrophysiological properties of a C-terminal domain truncation of the Mycobacterium tuberculosis MscL (MtMscLΔC). A crystal structure of MtMscLΔC solubilized in the detergent n-dodecyl-β-D-maltopyranoside reveals the pentameric, closed state-like architecture for the membrane spanning region observed in the previously solved full-length MtMscL. Electrophysiological characterization demonstrates that MtMscLΔC retains mechanosensitivity, but with conductance and tension sensitivity more closely resembling full length EcMscL than MtMscL. This study establishes that the C-terminal domain of MtMscL is not required for oligomerization of the full-length channel, but rather influences the tension sensitivity and conductance properties of the channel. The collective picture that emerges from these data is that each MscL channel structure has characteristic features, highlighting the importance of studying multiple homologs.

Cryo-EM structure of 5-HT3A receptor in its resting conformation

Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating.

Functional Analysis of TRPV1 Channels with a Genetically Encoded Cross-Linker

TRP channels constitute a family of non-selective, polymodal cation channels. They can sense different nature stimulus, i.e. chemical, electric, thermic and mechanic. The members of this family are homologous to the voltage-gated cation channels superfamily. Consequently, TRP channels have a tetrameric structure, composed by four monomers with six transmembrane domains (TMD) each, and substantial N- and C-terminal intracellular domains. The S1-S4 TMD region is considered the ligand binding domain, and the pore of the channel is comprised by the S5-S6 TMD and a pore loop between them. High-resolution structures are now available from multiple members of the TRPV subfamily. These structures reveal rich interaction networks between the N- and C-terminal domains, the intracellular loops and the pore forming helixes. For instance, the TRP domain, which is formed by an extended alpha-helix from the sixth TMD, is localized in the core of this interaction network, thus serving as a putative signaling integrator between modulatory signals and the channels gating mechanism(s). In order gather site- and conformation-specific information on TRPV1 function, we applied nonsense suppression to encode the non-canonical amino acid p-Benzoylphenylalanine (BPA) into conserved aromatic sites into TRPV1 in HEK cells. This aromatic amino acid is useful for functional perturbation analysis studies and in voltage-clamp UV dependent crosslinking assays. Specific encoding of BPA was achieved with an evolved E. coli. tyrosine synthetase and tRNA. Full length channel rescue at introduced TAG sites in the presence of BPA was first demonstrated by western blot of cell lysates. Functional expression was confirmed with whole-cell voltage clamp of TRPV1 channels in HEK293 cells, demonstrating that BPA containing channels could be gated by temperature and/or capsaicin induced activation. These data set the stage for site-specific cross-linking and the potential capture of transient gating states.

A Novel Form of CaV1.4 Ca2+ Channel Regulation Revealed by Alternative Splicing and a Mutation Causing Congenital Stationary Night Blindness

To support its role in mediating tonic exocytosis at the photoreceptor synapse, Cav1.4 L- type Ca2+ channels undergo weak Ca2+-dependent inactivation (CDI). The mechanism involves a C-terminal automodulatory domain (CTM) that competes with calmodulin (CaM) binding to the channel. A mutation that causes congenital stationary night blindness (CSNB2), K1591X, causes premature truncation of the channel just downstream of the CaM binding region and removal of the entire CTM. The mutant channels (Cav1.4K1591X) exhibit strong CDI and activation at more negative voltages than full-length channels similar to a splice variant (Cav1.4Δex47) that lacks just a portion of the CTM. To determine if selective deletion of exon 47 causes distinct regulation of CDI and activation as compared to the K1591X mutation, we compared the properties of Cav1.4K1591X and Cav1.4Δex47 in electrophysiological recordings of transfected HEK293T cells. We found key differences in how CaM regulates CDI of these channels. First, Cav1.4K1591X undergoes significantly faster CDI than Cav1.4Δex47. Second, CDI of Cav1.4Δex47 can be suppressed by dominant negative expression of CaM mutants that cannot bind Ca2+ in either the C-terminal (CaM34) or N-terminal (CaM12) lobe, whereas only CaM34 blunts CDI of Cav1.4K1591X. Third, CaM12 reverses the negative shift in activation of Cav1.4Δex47 but not of Cav1.4K1591X. We conclude that the deletion of exon 47 alters how CaM functionally interacts with the channel in a way that is not reproduced by the K1591X mutation, which may contribute to the pathological effects of K1591X for Ca2+ signaling at the photoreceptor synapse.

Readthrough of SCN5A Nonsense Mutations p.R1623X and p.S1812X Questions Gene-therapy in Brugada Syndrome.

Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression. HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.

Full length channel pressure tube sagging study under postulated LOCA with un-availability of ECCS in an Indian PHWR

An experimental investigation consisting of two experiments was conducted to assess the thermal behavior of the Indian-made full length Pressure Tube (PT) of an Indian PHWR. Loss of Coolant Accident (LOCA) with Emergency Core Cooling System (ECCS) failure postulated scenario was simulated by resistance heating the pressure tube to generate heat equal to the decay heat of nuclear fuel under such scenario after shutdown. On heating, the pressure tube deformed by sagging and eventually contacted the Calandria Tube (CT). After first contact, the temperature rise was arrested near the contact region but locations far away from the first contact continued to get heated up. The second contact, however, arrested the PT temperature rise completely, hence safeguarding the PT integrity and establishing moderator as an effective heat sink. The initiation of sagging occurred at around 460°C. The PT-CT contact took place at a contact temperature of 665–669°C at the bottom of the PT.

Kv2.1 Clustering Contributes to Insulin Exocytosis and Rescues Human β-Cell Dysfunction.

Insulin exocytosis is regulated by ion channels that control excitability and Ca2+ influx. Channels also play an increasingly appreciated role in microdomain structure. In this study, we examine the mechanism by which the voltage-dependent K+ (Kv) channel Kv2.1 (KCNB1) facilitates depolarization-induced exocytosis in INS 832/13 cells and β-cells from human donors with and without type 2 diabetes (T2D). We find that Kv2.1, but not Kv2.2 (KCNB2), forms clusters of 6-12 tetrameric channels at the plasma membrane and facilitates insulin exocytosis. Knockdown of Kv2.1 expression reduces secretory granule targeting to the plasma membrane. Expression of the full-length channel (Kv2.1-wild-type) supports the glucose-dependent recruitment of secretory granules. However, a truncated channel (Kv2.1-ΔC318) that retains electrical function and syntaxin 1A binding, but lacks the ability to form clusters, does not enhance granule recruitment or exocytosis. Expression of KCNB1 appears reduced in T2D islets, and further knockdown of KCNB1 does not inhibit Kv current in T2D β-cells. Upregulation of Kv2.1-wild-type, but not Kv2.1-ΔC318, rescues the exocytotic phenotype in T2D β-cells and increases insulin secretion from T2D islets. Thus, the ability of Kv2.1 to directly facilitate insulin exocytosis depends on channel clustering. Loss of this structural role for the channel might contribute to impaired insulin secretion in diabetes.