Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca²⁺- and voltage-activated K⁺ (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC₅₀ for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10⁶ M⁻¹s⁻¹. Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity.     

Direct observation of a preinactivated, open state in BK channels with beta2 subunits

Proteins arising from the Slo family assemble into homotetramers to form functional large-conductance, Ca2+- and voltage-activated K+ channels, or BK channels. These channels are also found in association with accessory beta subunits, which modulate several aspects of channel gating and expression. Coexpression with either of two such subunits, beta2 or beta3b, confers time-dependent inactivation onto BK currents. mSlo1+beta3b channels display inactivation that is very rapid but incomplete. Previous studies involving macroscopic recordings from these channels have argued for the existence of a second, short-lived conducting state in rapid equilibrium with the nonconducting, inactivated conformation. This state has been termed "pre-inactivated," or O*. beta2-mediated inactivation, in contrast, occurs more slowly but is virtually complete at steady state. Here we demonstrate, using both macroscopic and single channel current recordings, that a preinactivated state is also a property of mSlo1+beta2 channels. Detection of this state is enhanced by a mutation (W4E) within the initial beta2 NH2-terminal segment critical for inactivation. This mutation increases the rate of recovery to the preinactivated open state, yielding macroscopic inactivation properties qualitatively more similar to those of beta3b. Furthermore, short-lived openings corresponding to entry into the preinactivated state can be observed directly with single-channel recording. By examining the initial openings after depolarization of a channel containing beta2-W4E, we show that channels can arrive directly at the preinactivated state without passing through the usual long-lived open conformation. This final result suggests that channel opening and inactivation are at least partly separable in this channel. Mechanistically, the preinactivated and inactivated conformations may correspond to binding of the beta subunit NH2 terminus in the vicinity of the cytoplasmic pore mouth, followed by definitive movement of the NH2 terminus into a position of occlusion within the ion-conducting pathway.     

Closed-channel block of BK potassium channels by bbTBA requires partial activation

Blockade of large-conductance Ca²⁺-activated K⁺ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca²⁺-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca²⁺ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca²⁺; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.     

Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform

Background

The c D NA-mediated A nnealing, extension, S election and L igation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel^v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.

Methods

Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t -statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).

Results

Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1.

Conclusions

The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.     

Nav1.3 and FGF14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.     

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice

Introduction

The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo.

Methods

Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system.

Results

OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation.

Conclusions

Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.     

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERβ and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERβ and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERβ antibodies are insensitive for ERβ and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERβ antibody is shown to be highly specific and sensitive for detection of full-length ERβ and its variant forms. Strong and variable staining patterns for endogenous levels of ERβ protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERβ was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERβ antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERβ in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERβ, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.     

Species-specific Differences among KCNMB3 BK beta3 auxiliary subunits: some beta3 N-terminal variants may be primate-specific subunits

The KCNMB3 gene encodes one of a family of four auxiliary beta subunits found in the mammalian genome that associate with Slo1 alpha subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct beta3 subunits, beta3a-d. Three variants, beta3a-c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse beta3 variants. Whereas beta1, beta2, and beta4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse beta3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, beta3a and beta3b, of the four found in humans. Both human and mouse beta3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hbeta3b exhibits rapid inactivation, the putative mbeta3b does not inactivate. Furthermore, unlike hbeta3, the mbeta3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca(2+) concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mbeta3b N terminus may not be a true orthologue of the hbeta3b N terminus and that both beta3c and beta3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous beta3 subunits may differ among mammalian species; second, the specific physiological roles of homologous beta3 subunits may differ among mammalian species; and, third, some beta3 variants may be primate-specific ion channel subunits.     

Membrane diffusion- and capillary blood volume measurements are not useful as screening tools for pulmonary arterial hypertension in systemic sclerosis: a case control study

Background

Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.

Methods

Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.

Results

Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.

Conclusion

Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.