Chloroplasts require protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to import thousands of cytoplasmically synthesized preproteins. However, the ...molecular identity of the TIC translocon remains controversial. Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. We purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. Thus, this complex constitutes a general TIC translocon.
Abstract
Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement ...efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.
KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we ...revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.
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•The electrostatic state of the cytoplasmic domain affects inactivation in KcsA channel.•A D149N mutation induces loss of fast inactivation and high K+ selectivity, which are characteristics of wild-type KcsA.•R153A mutation prevents the loss of fast inactivation induced by D149N.
Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, ...Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.
Cry4Aa toxin produced by
Bacillus thuringiensis
subsp.
israelensis
exhibits specific toxicity to larvae of medically important mosquito genera. In the present study, we analyzed the characteristics ...of channel pores formed by recombinant Cry4Aa in a solvent-free planar lipid bilayer. Stable channel currents were observed in electrophysiologic measurements, and the single-channel conductance was 187 ± 10 pS in symmetrical buffer containing 150 mM KCl. The channel pores formed by Cry4Aa were cation-selective, with an estimated
P
K
/
P
Cl
permeability ratio of 4.9. In addition, Cry4Aa channel pores exhibited apparent cation preference in the order Na
+
> K
+
, Na
+
> Ca
2+
, and K
+
> Ca
2+
. Although the effect was limited, the cation preference of Cry4Aa channel pores seemed to be correlated with toxicity.
Culex pipiens
mosquito larvae reared in NaCl solution exhibited greater sensitivity to Cry4Aa, particularly early period after exposure. The presence of cations that preferentially translocate through Cry4Aa channel pores may facilitate excessive influx of water into the midgut cells, leading to colloid-osmotic lysis. Whereas CaCl
2
had some effect on the mosquito-larvicidal activity of Cry4Aa, KCl had no effect. The effect of some cations may be mitigated by the variety of ion channels present on the midgut cell membrane.
Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for ...the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.
Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the ...channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.
•Chronic kidney disease, inferior mesenteric artery (IMA), and lumbar arteries were risk factors for aneurysm sac enlargement.•Patent IMA seemed to have the greatest impact on long-term aneurysm sac ...enlargement.•Prognosis of patient with occluded IMA was quite good.
Although the preoperative risk factors associated with the occurrence of type II endoleak (ETII) after endovascular aortic repair (EVAR) have gradually become more evident, the preoperative risk factors associated with aneurysm sac enlargement caused by ETII remain unclear. This study aimed to determine the preoperative risk factors associated with aneurysm sac enlargement caused by ETII after EVAR.
This retrospective cohort study reviewed 519 EVARs performed for true abdominal aortic aneurysm between January 2006 and December 2018 at our institution. EVARs using commercially available bifurcated devices with no type I or III endoleaks during follow-up and with ≥12 months follow-up were included. A total of 320 patients were enrolled in the study. To identify the preoperative risk factors of sac enlargement after EVAR, Cox regression analysis was used to assess preoperative data.
The median follow-up period was 60.8 months. Overall, 135 of 320 patients (42%) had ETII during follow-up, and 47 of 135 patients (35%) developed aneurysm sac enlargement. Multivariate analysis revealed that chronic kidney disease (CKD) stage ≥4 (hazard ratio HR, 4.65; 95% confidence interval CI, 2.13–10.15; P = 0.001), patent inferior mesenteric artery (IMA) (HR, 17.85; 95% CI, 2.46–129.73; P< 0.001), and number of patent lumbar arteries (LAs) (HR, 1.37; 95% CI, 1.13–1.68; P= 0.002) were risk factors of aneurysm sac enlargement caused by ETII.
CKD stage ≥4, patent IMA, and number of patent LAs were independent risk factors for aneurysm sac enlargement after EVAR. In particular, patent IMA had the highest HR and seemed to have the greatest impact on long-term aneurysm sac enlargement. Hence, taking preoperative measures to address a patent IMA appears to be important in reducing the incidence of sac enlargement.
The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of ...cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.
Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and ...Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.
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