Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22 (TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

A Cloned Gene HuBADH from Hylocereus undatus Enhanced Salt Stress Tolerance in Transgenic Arabidopsis thaliana Plants.

BACKGROUND: Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine and is considered to be a type of osmoregulator, so it can play a role in plants' responses to abiotic stresses. METHODS: In this study, a novel HuBADH gene from Hylocereus undatus (pitaya) was cloned, identified, and sequenced. The full-length cDNA included a 1512 bp open reading frame that encoded a 54.17 kDa protein consisting of 503 amino acids. Four oxidation-related stress-responsive marker genes (FSD1, CSD1, CAT1, and APX2) were analyzed by Quantitative real-time reverse transcription (qRT-PCR) in wild type (WT) and transgenic A. thaiana overexpression lines under NaCl stress. RESULTS: HuBADH showed high homology (79-92%) with BADH of several plants. The HuBADH gene was genetically transformed into Arabidopsis thaliana and overexpressed in transgenic lines, which accumulated less reactive oxygen species than WT plants, and had higher activities of antioxidant enzymes under NaCl stress (i.e., 300 mM). All four marker genes were significantly upregulated in WT and HuBADH-overexpressing transgenic A. thaliana plants under salt stress. Glycine betaine (GB) content was 32-36% higher in transgenic A. thaliana lines than in WT in the control (70-80% in NaCl stress). CONCLUSIONS: Our research indicates that HuBADH in pitaya plays a positive modulatory role when plants are under salt stress.

Clinicopathological characteristics, treatment and prognosis of oral adenocarcinoma: a population-based study.

OBJECTIVES: The aim of this study was to identify clinicopathologic features, treatment and prognosis of oral adenocarcinoma (OADC). STUDY DESIGN: Retrospective cohort analysis. SETTING: National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) program. METHODS: Patients diagnosed with OADC between 2000 to 2018 were identified from the SEER database. Overall survival (OS) and disease-specific survival (DSS) were assessed using Kaplan-Meier analyses and Cox regression models. RESULTS: There were 924 OADC and 37,500 oral squamous cell carcinoma (OSCC) patients identified. Patients with OADC were more significantly associated with younger age, female gender, well differentiation and early AJCC Clinical stage. The study revealed that patients with OADC had better 10-year OS and DSS than those with OSCC (OS: 69.3% vs 40.8%, P < 0.001; DSS: 83.6% vs 53.3%, P < 0.001). The survival advantage still persisted in multivariable analyses (OS: hazard ratio [HR] = 0.427, P < 0.001; DSS: HR = 0.320, P < 0.001). For OADC, multivariable analysis showed that advanced age, stage, and histologic grade were associated with worse OS and DSS, and surgery was associated with better OS and DSS. CONCLUSIONS: OADC has a significantly better prognosis than OSCC, with better differentiation, and more early stage. Surgery was the preferred treatment, for patients with lymph node metastasis, radiotherapy may afford a survival benefit.

Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

OBJECTIVES: To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. METHODS: We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a T cell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed. RESULTS: Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. CONCLUSION: Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Assessment of heterosis based on parental genetic distance estimated with SSR and SNP markers in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Heterosis has been extensively utilized in different crops and made a significant contribution to global food security. Genetic distance (GD) is one of the valuable criteria for selecting parents in hybrid breeding. The objectives of this study were to estimate the GD between parents using both simple sequence repeat (SSR) markers and single nucleotide polymorphism (SNP) markers and to investigate the efficiency of the prediction of hybrid performance based on GD. The experiment comprised of four male parents, 282 female parents and 1128 F1, derived from NCII mating scheme. The hybrids, their parents and two check cultivars were evaluated for two years. Performance of F1, mid-parent heterosis (MPH), and best parent heterosis (BPH) were evaluated for ten agronomic and fiber quality traits, including plant height, boll weight, boll number, lint percentage, fiber length, fiber strength, fiber uniformity, fiber elongation ratio, micronaire, and spinning consistent index. RESULTS: Heterosis was observed in all hybrids and, the traits like plant height, boll number, boll weight and lint percentage exhibited higher heterosis than the fiber quality traits. Correlations were significant between parental and F1 performances. The F1 performances between three hybrid sets (Elite×Elite, Exotic×Elite, and Historic×Elite) showed significant differences in eight traits, including boll number, lint percentage, fiber length, fiber strength, fiber uniformity, fiber elongation ratio, micronaire, and spinning consistent index. The correlation of the GD assessed by both SSR and SNP markers was significantly positive. The cluster analysis based on GD results estimated using SNP showed that all the female parents divided into five groups and the F1 performance between these five groups showed significant differences in four traits, including lint percentage, micronaire, fiber strength, and fiber elongation ratio. The correlation between GD and F1 performance, MPH and BPH were significant for lint percentage and micronaire. CONCLUSIONS: Our results suggested that GD between parents could be helpful in heterosis prediction for certain traits. This study reveals that molecular marker analysis can serve as a basis for assigning germplasm into heterotic groups and to provide guidelines for parental selection in hybrid cotton breeding.

ILT3 (LILRB4) Promotes the Immunosuppressive Function of Tumor-Educated Human Monocytic Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. IMPLICATIONS: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Genome-wide dissection of hybridization for fiber quality- and yield-related traits in upland cotton.

An evaluation of combining ability can facilitate the selection of suitable parents and superior F1 hybrids for hybrid cotton breeding, although the molecular genetic basis of combining ability has not been fully characterized. In the present study, 282 female parents were crossed with four male parents in accordance with the North Carolina II mating scheme to generate 1128 hybrids. The parental lines were genotyped based on restriction site-associated DNA sequencing and 306 814 filtered single nucleotide polymorphisms were used for genome-wide association analysis involving the phenotypes, general combining ability (GCA) values, and specific combining ability values of eight fiber quality- and yield-related traits. The main results were: (i) all parents could be clustered into five subgroups based on population structure analyses and the GCA performance of the female parents had significant differences between subgroups; (ii) 20 accessions with a top 5% GCA value for more than one trait were identified as elite parents for hybrid cotton breeding; (iii) 120 significant single nucleotide polymorphisms, clustered into 66 quantitative trait loci, such as the previously reported Gh_A07G1769 and GhHOX3 genes, were found to be significantly associated with GCA; and (iv) identified quantitative trait loci for GCA had a cumulative effect on GCA of the accessions. Overall, our results suggest that pyramiding the favorable loci for GCA may improve the efficiency of hybrid cotton breeding.

An AP2/ERF Gene, HuERF1, from Pitaya (Hylocereus undatus) Positively Regulates Salt Tolerance.

Pitaya (Hylocereus undatus) is a high salt-tolerant fruit, and ethylene response factors (ERFs) play important roles in transcription-regulating abiotic tolerance. To clarify the function of HuERF1 in the salt tolerance of pitaya, HuERF1 was heterogeneously expressed in Arabidopsis. HuERF1 had nuclear localization when HuERF1::GFP was expressed in Arabidopsis protoplasts and had transactivation activity when HuERF1 was expressed in yeast. The expression of HuERF1 in pitaya seedlings was significantly induced after exposure to ethylene and high salinity. Overexpression of HuERF1 in Arabidopsis conferred enhanced tolerance to salt stress, reduced the accumulation of superoxide (O2∙) and hydrogen peroxide (H2O2), and improved antioxidant enzyme activities. These results indicate that HuERF1 is involved in ethylene-mediated salt stress tolerance, which may contribute to the salt tolerance of pitaya.

Insights into the Evolution of Neoteny from the Genome of the Asian Icefish Protosalanx chinensis.

Salangids, known as Asian icefishes, represent a peculiar radiation within the bony fish order Protacanthopterygii where adult fish retain larval characteristics such as transparent and miniaturized bodies and a cartilaginous endoskeleton into adulthood. Here, we report a de novo genome of Protosalanx chinensis, the most widely distributed salangid lineage. The P. chinensis genome assembly is more contiguous and complete than a previous assembly. We estimate that P. chinensis, salmons, trouts, and pikes diverged from a common ancestor 185 million years ago. A juxtaposition with other fish genomes revealed loss of the genes encoding ectodysplasin-A receptor (EDAR), SCPP1, and four Hox proteins and likely lack of canonical fibroblast growth factor 5 (FGF5) function. We also report genomic variations of P. chinensis possibly reflecting the immune system repertoire of a species with a larval phenotype in sexually mature individuals. The new Asian icefish reference genome provides a solid foundation for future studies.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Integration of conventional and advanced molecular tools to track footprints of heterosis in cotton.

BACKGROUND: Heterosis, a multigenic complex trait extrapolated as sum total of many phenotypic features, is widely utilized phenomenon in agricultural crops for about a century. It is mainly focused on establishing vigorous cultivars with the fact that its deployment in crops necessitates the perspective of genomic impressions on prior selection for metric traits. In spite of extensive investigations, the actual mysterious genetic basis of heterosis is yet to unravel. Contemporary crop breeding is aimed at enhanced crop production overcoming former achievements. Leading cotton improvement programs remained handicapped to attain significant accomplishments. RESULTS: In mentioned context, a comprehensive project was designed involving a large collection of cotton accessions including 284 lines, 5 testers along with their respective F1 hybrids derived from Line × Tester mating design were evaluated under 10 diverse environments. Heterosis, GCA and SCA were estimated from morphological and fiber quality traits by L × T analysis. For the exploration of elite marker alleles related to heterosis and to provide the material carrying such multiple alleles the mentioned three dependent variables along with trait phenotype values were executed for association study aided by microsatellites in mixed linear model based on population structure and linkage disequilibrium analysis. Highly significant 46 microsatellites were discovered in association with the fiber and yield related traits under study. It was observed that two-thirds of the highly significant associated microsatellites related to fiber quality were distributed on D sub-genome, including some with pleiotropic effect. Newly discovered 32 hQTLs related to fiber quality traits are one of prominent findings from current study. A set of 96 exclusively favorable alleles were discovered and C tester (A971Bt) posited a major contributor of these alleles primarily associated with fiber quality. CONCLUSIONS: Hence, to uncover hidden facts lying within heterosis phenomenon, discovery of additional hQTLs is required to improve fibre quality. To grab prominent improvement in influenced fiber quality and yield traits, we suggest the A971 Bt cotton cultivar as fundamental element in advance breeding programs as a parent of choice.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

Oral treatment with a gamma-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease.

The beta-amyloid peptide (Abeta) is thought to play a critical role in the pathophysiology of Alzheimer's disease (AD). To study the effects of Abeta on the brain, transgenic mouse models have been developed that express high levels of Abeta. These mice show some features of AD, including amyloid plaques and mild cognitive impairment, but not others such as progressive neurodegeneration. We investigated the age-dependent effects of Abeta on synaptic physiology in Tg2576 mice that express human Abeta. We report that both basal synaptic activity and long-term potentiation (LTP), as measured in the CA1 region of the hippocampus, were compromised by 7 months of age before plaque deposition. Despite a persistent increase in Abeta levels with age, LTP recovered in 14-month-old mice, with no further loss of basal activity compared with activity measured in 7-month-old mice. Previous work has shown that inhibitors of gamma-secretase, an enzyme critical for Abeta synthesis, can significantly reduce Abeta production and plaque formation in Tg2576 mice. Our data demonstrate that 7-month-old Tg2576 mice treated with an orally available gamma-secretase inhibitor showed a significant improvement in synaptic function and plasticity within days, and the effect was correlated with the extent and duration of Abeta reduction. These results indicate that recovery from Abeta-mediated synaptotoxicity can occur rapidly with Abeta-lowering therapies. These findings highlight some of the strengths and limitations of using Abeta-overexpressing mouse models for Alzheimer's drug discovery.

W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition.

Ca(+)-calmodulin (Ca(2+)-CaM)-dependent protein kinase II (Ca(2+)/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca(2+)-occupied CaM, and KN-93, an inhibitor of Ca(2+)/CaMKII, on the K(v)4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC(50) of 92.4 muM. The block was voltage dependent, with an effective electrical distance of 0.18 +/- 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of K(v)4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 muM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated K(v)4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 muM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca(2+)/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca(2+)-CaM, as well as a change in activity of Ca(2+)-CaM-dependent enzymes, including Ca(2+)/CaMKII.

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4.

Voltage-gated K(+) channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K(+) currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At -60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K(+) channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K(+) channels.

Time- and voltage-dependent components of Kv4.3 inactivation.

Kv4.3 inactivation is a complex multiexponential process, which can occur from both closed and open states. The fast component of inactivation is modulated by the N-terminus, but the mechanisms mediating the other components of inactivation are controversial. We studied inactivation of Kv4.3 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Inactivation during 2000 ms pulses at potentials positive to the activation threshold was described by three exponents (46 +/- 3, 152 +/- 13, and 930 +/- 50 ms at +50 mV, n = 7) whereas closed-state inactivation (at potentials below threshold) was described by two exponents (1079 +/- 119 and 3719 +/- 307 ms at -40 mV, n = 9). The fast component of open-state inactivation was dominant at potentials positive to -20 mV. Negative to -30 mV, the intermediate and slow components dominated inactivation. Inactivation properties were dependent on pulse duration. Recovery from inactivation was strongly dependent on voltage and pulse duration. We developed an 11-state Markov model of Kv4.3 gating that incorporated a direct transition from the open-inactivated state to the closed-inactivated state. Simulations with this model reproduced open- and closed-state inactivation, isochronal inactivation relationships, and reopening currents. Our data suggest that inactivation can proceed primarily from the open state and that multiple inactivation components can be identified.

Activation properties of Kv4.3 channels: time, voltage and [K+]o dependence.

Rapidly inactivating, voltage-dependent K(+) currents play important roles in both neurones and cardiac myocytes. Kv4 channels form the basis of these currents in many neurones and cardiac myocytes and their mechanism of inactivation appears to differ significantly from that reported for Shaker and Kv1.4 channels. In most channel gating models, inactivation is coupled to the kinetics of activation. Hence, there is a need for a rigorous model based on comprehensive experimental data on Kv4.3 channel activation. To develop a gating model of Kv4.3 channel activation, we studied the properties of Kv4.3 channels in Xenopus oocytes, without endogenous KChIP2 ancillary subunits, using the perforated cut-open oocyte voltage clamp and two-electrode voltage clamp techniques. We obtained high-frequency resolution measurements of the activation and deactivation properties of Kv4.3 channels. Activation was sigmoid and well described by a fourth power exponential function. The voltage dependence of the activation time constants was best described by a biexponential function corresponding to at least two different equivalent charges for activation. Deactivation kinetics are voltage dependent and monoexponential. In contrast to other voltage-sensitive K(+) channels such as HERG and Shaker, we found that elevated extracellular [K(+)] modulated the activation process by slowing deactivation and stabilizing the open state. Using these data we developed a model with five closed states and voltage-dependent transitions between the first four closed states coupled to a voltage-insensitive transition between the final closed (partially activated) state and the open state. Our model closely simulates steady-state and kinetic activation and deactivation data.

Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect.

We studied quinidine block of Kv1.4DeltaN, a K(+) channel lacking N-type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non-additively) by interventions that slow C-type inactivation: [K(+)](o) elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ~10-fold, but left the effective electrical distance unchanged at ~0.15. Following rapid open channel block, there were time-dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time-dependent aspect of quinidine block was similar to recovery from normal C-type inactivation. Manipulations that prevented the channel from entering the C-type inactivated state (i.e. high [K(+)](o) or the K532Y mutation) prevented the development of the time-dependent quinidine-induced inactivation. The concentration dependence of the rapid block and the time-dependent quinidine-induced inactivation were similar, but the time-dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage-sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C-type inactivated state in a time- and voltage-dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C-type inactivated state through a voltage-dependent conformational change.

Kinetic properties of Kv4.3 and their modulation by KChIP2b.

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.