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1.
Polyunsaturated fatty acids (PUFAs), but not saturated fatty acids, modulate ion channels such as the cardiac KCNQ1 channel, although the mechanism is not completely understood. Using both simulations and experiments, we find that PUFAs interact directly with the KCNQ1 channel via two different binding sites: one at the voltage sensor and one at the pore. These two amphiphilic binding pockets stabilize the negatively charged PUFA head group by electrostatic interactions with R218, R221, and K316, while the hydrophobic PUFA tail is selectively stabilized by cassettes of hydrophobic residues. The rigid saturated tail of stearic acid prevents close contacts with KCNQ1. By contrast, the mobile tail of PUFA linoleic acid can be accommodated in the crevice of the hydrophobic cassette, a defining feature of PUFA selectivity in KCNQ1. In addition, we identify Y268 as a critical PUFA anchor point underlying fatty acid selectivity. Combined, this study provides molecular models of direct interactions between PUFAs and KCNQ1 and identifies selectivity mechanisms. Long term, this understanding may open new avenues for drug development based on PUFA mechanisms.  相似文献   

2.
《Biophysical journal》2021,120(20):4429-4441
The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.  相似文献   

3.
Polyunsaturated fatty acids (PUFAs) have beneficial effects on epileptic seizures and cardiac arrhythmia. We report that ω-3 and ω-6 all-cis-PUFAs affected the voltage dependence of the Shaker K channel by shifting the conductance versus voltage and the gating charge versus voltage curves in negative direction along the voltage axis. Uncharged methyl esters of the PUFAs did not affect the voltage dependence, whereas changes of pH and charge mutations on the channel surface affected the size of the shifts. This suggests an electrostatic effect on the channel's voltage sensors. Monounsaturated and saturated fatty acids, as well as trans-PUFAs did not affect the voltage dependence. This suggests that fatty acid tails with two or more cis double bonds are required to place the negative carboxylate charge of the PUFA in a position to affect the channel's voltage dependence. We propose that charged lipophilic compounds could play a role in regulating neuronal excitability by electrostatically affecting the channel's voltage sensor. We believe this provides a new approach for pharmacological treatment that is voltage sensor pharmacology.  相似文献   

4.
Many proteins function by changing conformation in response to ligand binding or changes in other factors in their environment. Any change in the sequence of a protein, for example during evolution, which alters the relative free energies of the different functional conformations changes the conditions under which the protein will function. Voltage-gated ion channels are membrane proteins that open and close an ion-selective pore in response to changes in transmembrane voltage. The charged S4 transmembrane helix transduces changes in transmembrane voltage into a change in protein internal energy by interacting with the rest of the channel protein through a combination of non-covalent interactions between adjacent helices and covalent interactions along the peptide backbone. However, the structural basis for the wide variation in the V50 value between different voltage-gated potassium channels is not well defined. To test the role of the loop linking the S3 helix and the S4 helix in voltage sensitivity, we have constructed a set of mutants of the rat Kv1.2 channel that vary solely in the length and composition of the extracellular loop that connects S4 to S3. We evaluated the effect of these different loop substitutions on the voltage sensitivity of the channel and compared these experimental results with molecular dynamics simulations of the loop structures. Here, we show that this loop has a significant role in setting the precise V50 of activation in Kv1 family channels.  相似文献   

5.
多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族KV7通道和大电导钙离子激活的钾离子通道(BKCa)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕KV7和BKCa通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大KV7和BKCa通道的电流幅值,其中对KV7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BKCa通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对KV7和BKCa通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对KV7和BKCa通道调节作用效果和分子机制,有助于全面理解KV7和BK  相似文献   

6.
A ketogenic diet is an alternative treatment of epilepsy in infants. The diet, rich in fat and low in carbohydrates, elevates the level of polyunsaturated fatty acids (PUFAs) in plasma. These substances have therefore been suggested to contribute to the anticonvulsive effect of the diet. PUFAs modulate the properties of a range of ion channels, including K and Na channels, and it has been hypothesized that these changes may be part of a mechanistic explanation of the ketogenic diet. Using computational modelling, we here study how experimentally observed PUFA-induced changes of ion channel activity affect neuronal excitability in CA1, in particular responses to synaptic input of high synchronicity. The PUFA effects were studied in two pathological models of cellular hyperexcitability associated with epileptogenesis. We found that experimentally derived PUFA modulation of the A-type K (KA) channel, but not the delayed-rectifier K channel, restored healthy excitability by selectively reducing the response to inputs of high synchronicity. We also found that PUFA modulation of the transient Na channel was effective in this respect if the channel''s steady-state inactivation was selectively affected. Furthermore, PUFA-induced hyperpolarization of the resting membrane potential was an effective approach to prevent hyperexcitability. When the combined effect of PUFA on the KA channel, the Na channel, and the resting membrane potential, was simulated, a lower concentration of PUFA was needed to restore healthy excitability. We therefore propose that one explanation of the beneficial effect of PUFAs lies in its simultaneous action on a range of ion-channel targets. Furthermore, this work suggests that a pharmacological cocktail acting on the voltage dependence of the Na-channel inactivation, the voltage dependences of KA channels, and the resting potential can be an effective treatment of epilepsy.  相似文献   

7.
Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.  相似文献   

8.
The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. KV2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in KV2.1. Y380 mutants do not introduce C-type inactivation into KV2.1 and have little effect on U-type inactivation of KV2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests KV2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, KV2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states.  相似文献   

9.
The atomic models of the Kv1.2 potassium channel in the active and resting state, originally presented elsewhere, are here refined using molecular dynamics simulations in an explicit membrane-solvent environment. With a minor adjustment of the orientation of the first arginine along the S4 segment, the total gating charge of the channel determined from >0.5 μs of molecular dynamics simulation is ∼12-12.7 e, in good accord with experimental estimates for the Shaker potassium channel, indicating that the final models offer a realistic depiction of voltage-gating. In the resting state of Kv1.2, the S4 segment in the voltage-sensing domain (VSD) spontaneously converts into a 310 helix over a stretch of 10 residues. The 310 helical conformation orients the gating arginines on S4 toward a water-filled crevice within the VSD and allows salt-bridge interactions with negatively charged residues along S2 and S3. Free energy calculations of the fractional transmembrane potential, acting upon key charged residues of the VSD, reveals that the applied field varies rapidly over a narrow region of 10-15 Å corresponding to the outer leaflet of the bilayer. The focused field allows the transfer of a large gating charge without translocation of S4 across the membrane.  相似文献   

10.
Auxiliary β-subunits dictate the physiological properties of voltage-gated K+ (KV) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific β-subunit of neuronal KV4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel’s voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated KV4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and KV1.3 channels or the KV4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the KV4.2-VSD is unique among the known actions of KV channel β-subunits. This study shows that the modular VSD of a KV channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic β-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.  相似文献   

11.
Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.  相似文献   

12.
The co-assembly of KCNQ1 with KCNE1 produces IKS, a K+ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K+ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.  相似文献   

13.
Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.  相似文献   

14.
The nature and magnitude of voltage sensor conformational changes during ion channel activation are controversial. We have analyzed the topology of the KVAP voltage sensor domain in the absence and presence of a hyperpolarized voltage using native, right-side out membrane vesicles from E. coli. This approach does not disrupt the normal membrane environment of the channel protein and does not involve detergent solubilization. We found that voltage-dependent conformational changes are focused in the N-terminal half of the KVAP S4 segment, in excellent agreement with results obtained with Shaker. Homologous residues in the KVAP and Shaker S4 segments are transferred from the extracellular to the intracellular compartment upon hyperpolarization. Taken together with X-ray structures indicating that the KVAP S4 segment is outwardly displaced at 0 mV compared to S4 in a mammalian Shaker channel, our results are consistent with the idea that S4 moves further during voltage-dependent activation in KVAP than in Shaker.  相似文献   

15.
Rapid inactivation of voltage-gated K+ (KV) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various KV channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different KV channels with hardly any sequence homology mediate quite similar effects even on unrelated KV channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution.  相似文献   

16.
Although crystal structures of various voltage-gated K+ (Kv) and Na+ channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K+ channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd2+ profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd2+ bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4–S1 Cd2+ bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.  相似文献   

17.
Kv channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.  相似文献   

18.
Kv channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10–20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker’s sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.  相似文献   

19.
Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the VSD from the voltage-dependent K+ channel KvAP (prokaryotic Kv from Aeropyrum pernix). The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. The differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid-body repositioning of the S3-S4 voltage-sensor paddle. Using 15N relaxation experiments, we show that much of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the picosecond-to-nanosecond timescale. In contrast, the kink in S3 is mobile on the microsecond-to-millisecond timescale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and showed that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.  相似文献   

20.
The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K+ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K+ channel, we have inserted a flag epitope in the NH2 terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K+ channel.  相似文献   

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