Modal behavior of the Kv1.1 channel conferred by the Kvβ1.1 subunit and its regulation by dephosphorylation of Kv1.1

Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kvbeta1.1 subunits (alpha(beta) channel) gives rise to a current that has a fast-inactivating and a sustained component; the proportion of the fast-inactivating component could be decreased by dephosphorylation of a basally phosphorylated Ser-446 on the alpha subunit. To account for our results we suggested a model that assumes a bimodal gating of the alpha(beta) channel. In this study, using single-channel analysis, we confirm this model. Two modes of gating were identified: (1) an inactivating mode characterized by low open probability and single openings early in the voltage step, and (2) a non-inactivating gating mode with bursts of openings. These two modes were non-randomly distributed, with spontaneous shifts between them. Each mode is characterized by a different set of open time constants (tau) and mean open times (t(0)). The non-inactivating mode is similar to the gating mode of a homomultimeric alpha channel. The phosphorylation-deficient alphaS446Abeta channel has the same two gating modes. Furthermore, alkaline phosphatase promoted the transition to the non-inactivating mode. This is the first report of modal behavior of a fast-inactivating K+ channel; furthermore, it substantiates the notion that direct phosphorylation is one mechanism that regulates the equilibrium between the two modes and thereby regulates the extent of macroscopic fast inactivation of a brain K+ channel.     

Modulation of Na+channel inactivation by the β1 subunit: A deletion analysis

Na⁺ currents recorded from Xenopus oocytes expressing the Na⁺ channel subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while coexpression with the ₁ subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (30-fold vs 5-fold). A re-assessment of steady-state inactivation, h (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the ₁ subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.     

Slow inactivation of the NaV1.4 sodium channel in mammalian cells is impeded by co-expression of the β1 subunit

In response to sustained depolarization or prolonged bursts of activity in spiking cells, sodium channels enter long-lived non-conducting states from which recovery at hyperpolarized potentials occurs over hundreds of milliseconds to seconds. The molecular basis for this slow inactivation remains unknown, although many functional domains of the channel have been implicated. Expression studies in Xenopus oocytes and mammalian cell lines have suggested a role for the accessory β1 subunit in slow inactivation, but the effects have been variable. We examined the effects of the β1 subunit on slow inactivation of skeletal muscle (NaV1.4) sodium channels expressed in HEK cells. Co-expression of the β1 subunit impeded slow inactivation elicited by a 30-s depolarization, such that the voltage dependence was right shifted (depolarized) and recovery was hastened. Mutational studies showed this effect was dependent upon the extracellular Ig-like domain, but was independent of the intracellular C-terminal tail. Furthermore, the β1 effect on slow inactivation was shown to be independent of the negative coupling between fast and slow inactivation.     

Modification of α subunit of RIIA sodium channels by aconitine

The effect of aconitine (AC), an alkaloid toxin, on the electrophysiological properties of the rat brain type IIA alpha subunit expressed heterologously in the Chinese Hamster Ovarian (CHO) cell line was studied under the whole-cell patch-clamp configuration. The activation threshold of modified channels shifted by about -40 mV. As the number of depolarizations increased, the transient current at 0 mV decreased and, in proportion, the AC-modified current at -50 mV increased. This suggests a transition of channels to an AC-modified state. The rate of modification was nearly four times faster when 50 microM AC was applied internally than when applied in the bath solution. This supports the idea that the AC-binding site is located close to the cytoplasmic mouth of the channel pore. The AC-modified sodium currents inactivated completely, although with slower kinetics. The steady-state inactivation followed a simple Boltzmann function. AC-modified currents activated without a sigmoidal delay. The permeability of the NH4+ ion was enhanced such that its permeability ratio increased from an initial value of 0. 18 to 0.95 and for Cs+ it was enhanced from 0.03 to 0.07. These studies show that the AC-binding site resides at the pore region of the alpha subunit of the Na+ channel, and that the presence of beta subunit/s is not essential for AC binding.     

Maspin expression is regulated by the non-canonical NF-κB subunit in androgen-insensitive prostate cancer cell lines

Dysregulation of Maspin expression and constitutive activation of NF-κB subunits are important events in tumorigenesis of prostate cancer. Recent finding points that RelB, which contributes to the alternative NF-κB activity, interferes with carcinogenesis in the prostate. We report here, that both the classical and the alternative NF-κB activities are constitutively present in androgen-insensitive human prostate cancer cells. Maspin and RelB expression is correlated negatively in prostate cancer tissues at the later stage. TNF-α signaling triggers the nuclear accumulation of RelB and the concomitant reduction of Maspin expression in a time-dependent manner. In addition, the proteasome inhibitor-induced Maspin expression is accompanied by the reduction of RelB expression. A successful depletion of RelB expression, but not RelA expression, induces Maspin expression. RelB-deficiency abrogates the proteasome inhibitor-induced Maspin expression. Moreover, we demonstrate that the enforced expression of RelB protein in prostate cancer cells inhibits Maspin expression. We propose that RelB is an essential molecule controlling the endogenous and the proteasome inhibitor-induced Maspin expression. Developing a RelB-targeted therapeutic intervention, which might be coupled with the induction of a tumor suppressor Maspin, is valuable in treating advanced, metastatic prostate cancer.     

Conformational change in the N-terminal domain of the Escherichia coli tryptophan synthase β2 subunit induced by its interactions with monoclonal antibodies

A fluorescence energy transfer signal was used to follow conformational changes occurring in two types of protein-protein complexes. The first complex studied was the native-like β₂ subunit of Escherichia coli tryptophan synthase reconstituted by reassembly of the N- and C-terminal proteolytic domains of the β chain. The other complexes were formed by the association of the N-terminal fragment (F₁) with a monoclonal antibody that recognizes the native dimeric protein; four such complexes, obtained with different antibodies that recognize four distinct antigenic sites on native β₂, were investigated. It was shown that a structural readjustment, which the isolated F₁ domain was unable to undergo alone, was imposed upon F₁ by interdomain interactions. Furthermore, with three of the four antibodies studied, the same conformational change in F₁ also occurred after formation of the F₁-antibody complex. These results demonstrate that, through an “induced fit mechanism”, antigen-antibody stereospecific assembly can force the polypeptide chain to adopt a structure more closely resembling the conformation it has in the native protein.     

Conformational change in the N-terminal domain of the Escherichia coli tryptophan synthase β2 subunit induced by its interactions with monoclonal antibodies

A fluorescence energy transfer signal was used to follow conformational changes occurring in two types of protein-protein complexes. The first complex studied was the native-like β₂ subunit of Escherichia coli tryptophan synthase reconstituted by reassembly of the N- and C-terminal proteolytic domains of the β chain. The other complexes were formed by the association of the N-terminal fragment (F₁) with a monoclonal antibody that recognizes the native dimeric protein; four such complexes, obtained with different antibodies that recognize four distinct antigenic sites on native β₂, were investigated. It was shown that a structural readjustment, which the isolated F₁ domain was unable to undergo alone, was imposed upon F₁ by interdomain interactions. Furthermore, with three of the four antibodies studied, the same conformational change in F₁ also occurred after formation of the F₁-antibody complex. These results demonstrate that, through an “induced fit mechanism”, antigen-antibody stereospecific assembly can force the polypeptide chain to adopt a structure more closely resembling the conformation it has in the native protein.     

PIKing the right isoform: the emergent role of the p110β subunit in breast cancer

Class IA phosphoinositide-3’-kinases (PI3Ks) regulate many cellular processes. Despite a clear implication of PI3K in cancer, the involvement of each of its isoforms namely p110α and p110β in the development of breast cancer remains elusive. Until recently, the spotlight was given to the α subunit; however, the p110β isoform has now emerged as an interesting target as well. In order to determine the importance of both these subunits in breast cancer, we aimed to study the expression of p110α and p110β in a series of invasive breast carcinomas. We constructed tissue microarrays from 315 invasive breast carcinomas and performed immunohistochemistry for p110α and β, correlating the expression patterns with clinicopathological parameters. Furthermore, overall survival was analysed through Kaplan–Meier survival curves and Cox regression. We found that p110 subunits are expressed in 23.8% of invasive breast carcinomas, of which 11.8% express p110α and 15.2% p110β. The p110α positive tumours correlated with hormone receptor (HR) expression, and were not associated with overall survival. The membrane expression of p110β was associated with worse prognosis. This was due to its link to HER2-overexpression, lower age of onset, higher grade, lymph node involvement, distant metastasis and was inversely associated with HR status. Furthermore, p110β expression was associated with worse overall survival. Importantly our results indicate a role for the beta subunit in the development/progression of HER2-overexpressing tumours, highlighting possible therapeutic associations between HER2 and p110β inhibitors.     

Immunohistochemical expression of the α5 integrin subunit in the normal adult rat central nervous system

We investigated the distribution of the 5 integrin subunit in the normal adult rat CNS using immunohistochemical methods. Results indicated that the 5 integrin subunit was expressed on the vast majority of neurons throughout the brain and spinal cord. In general, neurons showed diffuse cytoplasmic labelling, although many cortical neurons in layers 4 and 5 did show punctate labelling on the cell surface. In addition, axons within the white matter of the brainstem and caudal CNS areas were labelled, with the most intense labelling seen within the white matter of the spinal cord. In addition, labelling of astrocytes was seen throughout white matter, with particularly heavy astrocyte labelling in the spinal cord. The widespread distribution of the 5 subunit suggests a general function for the 51 integrin receptor (the only integrin receptor that includes the 5 subunit) in the adult CNS. The increased expression of fibronectin, the only known ligand for the 51 integrin receptor, known to occur around the site of a CNS lesion suggests a possible role for the 51 receptor in the response of neurons in the vicinity of a CNS injury.     

Regulation of the L-type calcium channel α-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines

The regulation of L-type voltage-dependent Ca²⁺ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive -1 subunits. PC12 cells showed, after prolonged exposure to a high-K⁺ depolarizing solution, a 25% reduction of the functional Ca²⁺ channel density which was accompanied by a decrease of the -1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, ⁴⁵Ca²⁺ uptake measurements revealed a fall in the functional activity of L-type Ca²⁺ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of -1 subunits allowed the recovery of the original density of L-type Ca²⁺ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca²⁺ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca²⁺ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca²⁺ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca²⁺ channels.     

Effects of the C-terminal Peptide of the αs subunit of the G protein on the regulation of Adenylyl Cyclase and Protein Kinase A activities by Biogenic Amines and Glucagon in Mollusk and Rat Muscles

The C-terminal parts of the subunits of heteromeric G proteins play an important role in the functional linkage of G proteins with receptors of the serpentine type. The present report describes studies of the effects of the C-terminal octapeptide 387–394 of the _s subunit of the mammalian G protein on the transmission of the hormonal signal via the hormone-sensitive adenylyl cyclase signal system, whose major components are receptors of the serpentine type, G proteins, and the enzymes adenylyl cyclase and protein kinase A. The peptide synthesized here, 387–394 amide (10^–7 - 10^–4 M), dose-dependently decreased adenylyl cyclase and protein kinase A activities stimulated by serotonin and glucagon in smooth muscle from the freshwater bivalve mollusk Anodonta cygnea and by the agonist isoproterenol in rat skeletal muscle. At a concentration as low as 10^–7 M, the peptide released potentiation of the stimulatory effects of hormones on adenylyl cyclase activity due to the non-hydrolyzable guanine nucleotide analog Gpp[NH]p. At the same time, it had almost no effect on the stimulation of adenylyl cyclase activity by non-hormonal agents (NaF, Gpp[NH]p, and forskolin). The inhibitory effects of hormones on adenylyl cyclase and protein kinase A activities persisted in the presence of the peptide. Our data demonstrate the importance of the C-terminal part of the _s subunit of the stimulatory G protein for its functional linkage with receptors of the serpentine type and throw light on the molecular mechanisms of the interactions between G proteins and receptors.Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 89, No. 7, pp. 837–850, July, 2003.     

Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its β1 subunit

Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I _NaL) for the function of both normal and, especially, failing myocardium, in which I _NaL is reportedly increased. While recent molecular studies identified the cardiac Na⁺ channel (NaCh) α subunit isoform (Na_v1.5) as a major contributor to I _NaL, the molecular mechanisms underlying alterations of I _NaL in heart failure (HF) are still unknown. Here we tested the hypothesis that I _NaL is modulated by the NaCh auxiliary β subunits. tsA201 cells were transfected simultaneously with human Na_v1.5 (former hH1a) and cardiac β₁ or β₂ subunits, and whole-cell patch-clamp experiments were performed. We found that I _NaL decay kinetics were significantly slower in cells expressing α + β₁ (time constant τ = 0.73 ± 0.16 s, n = 14, mean ± SEM, P < 0.05) but remained unchanged in cells expressing α + β₂ (τ = 0.52 ± 0.09 s, n = 5), compared with cells expressing Na_v1.5 alone (τ = 0.54 ± 0.09 s, n = 20). Also, β₁, but not β₂, dramatically increased I _NaL relative to the maximum peak current, I _NaT (2.3 ± 0.48%, n = 14 vs. 0.48 ± 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary β₁ subunit modulates I _NaL, produced by the human cardiac Na⁺ channel Na_v1.5 by slowing its decay and increasing I _NaL amplitude relative to I _NaT. Because expression of Na_v1.5 reportedly decreases but β₁ remains unchanged in chronic HF, the relatively higher expression of β₁ may contribute to the known I _NaL increase in HF via the modulation mechanism found in this study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Absence of F1-ATPase activity in Kluyveromyces lactis lacking the ɛ subunit

The mitochondrial F₁-ATPase is a multimeric enzyme, comprised of 3α, 3β, γ, δ and ɛ subunits, that is primarily responsible for the synthesis of ATP in eukaryotic cells. Recent work has shown that the F₁ complex of the petite-negative yeast Kluyveromyces lactis, with specific mutations in the α, β or γ subunits, has a novel function that suppresses lethality caused by loss of mtDNA. Previously, genes for the four largest subunits of K. lactis F₁ have been identified and characterised. In this study the gene coding for the ɛ-subunit of F₁, KlATPɛ, has been isolated and found to encode a polypeptide of 61 amino acids with only 32 residues identical to those in the protein from Saccharomyces cerevisiae. Strains carrying a null mutation of KlATPɛ are respiratory deficient while the introduction of ATPɛ from S. cerevisiae restores growth on non-fermentable carbon sources. In contrast to S. cerevisiae, K. lactis disrupted in ATPɛ does not have a detectable F₁-related mitochondrial ATP hydrolysis activity, suggesting that the ɛ-subunit plays a critical role in the formation of the catalytic sector of F₁. With a disrupted KlATPɛ, the ρ^o-lethality suppressor function of F₁ carrying the atp2-1 and atp1-6 alleles is abolished. However, inactivation of the ɛ subunit does not eliminate the ρ^o-viable phenotype of the atp1-1, atp2-9, atp3-2 mutants. It is suggested that the absence of ɛ may effect the assembly or stability of F₁ in the wild-type, atp 2-1 and atp1-6 strains, whereas the defect can be suppressed by the atp1-1, atp2-9 and atp3-2 mutations in the α, β and γ subunits respectively. Received: 9 November 1999 / 11 February 2000     

Ontogeny of thymic cortical epithelial cells expressing the thymoproteasome subunit β5t

Proteasomes are responsible for generating peptides presented by class I MHC molecules of the immune system. β5t, a recently identified proteasome component, is specifically expressed in thymic cortical epithelial cells (cTECs) and plays a pivotal role in generating an immunocompetent repertoire of class I MHC-restricted CD8(+) T cells. Here, we report that β5t is detectable in the thymus as early as E12.5 mouse embryos. We also found that β5t expression in cTECs was detectable in mice deficient for RelB or Rag2, indicating that β5t in cTECs is expressed in the absence of thymic medulla formation or thymocyte development beyond the CD4(-) CD8(-) stage. β5t expression in the embryonic thymus was not detectable in Foxn1-deficient nude mice, although its expression was not reduced in mice deficient for both CCR7 and CCR9, in which fetal thymus colonization by leukocytes is defective. These results indicate that β5t expression in cTECs is dependent on Foxn1 but independent of thymocyte crosstalk or thymic medulla formation.     

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit of the GABAA receptor

A line of mice was recently created in which the gabrb3 gene, which encodes the β₃ subunit of the GABA_A receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA_A receptors in the CNS enabled an investigation of the role of GABA and GABA_A receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA_A and GABA_B receptor agonists. Homozygous null (β₃^−/−) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (β₃^+/+) and heterozygous (β₃^+/−) littermates. The β₃^−/− mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their β₃^+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in β₃^−/− mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA_A receptors in the spinal cord. As expected, subcutaneous administration of the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in β₃^−/− mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, β₃^+/+ and β₃^+/− mice. However, the antinociceptive effect of the GABA_B receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in β₃^−/− mice compared to the progenitor strains, β₃^+/+ or β₃^+/− mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA_A receptors can affect the production of antinociception by other analgesic drugs as well.     

Evolutionary analysis of the plastid-encoded gene for the α subunit of the DNA-dependent RNA polymerase of Pyrenomonas salina (cryptophyceae)

The nucleotide sequence of the gene coding for the plastid-encoded subunit of DNA-dependent RNA polymerase from the cryptomonad alga Pyrenomonas salina was determined. The deduced amino-acid sequence, corresponding to a 35.2 kDa polypeptide, was compared to homologues from other organisms. Evolutionary relationships were analyzed in detail by the parsimony method together with bootstrap analysis. The deduced phylogenetic tree shows that the cryptomonad gene is the most ancient type of known plastid-encoded RNA polymerase.