Structural and mutational analysis of KCNQ2, the major gene locus for benign familial neonatal convulsions

Mutations in the voltage-gated potassium channel gene KCNQ2 on chromosome 20q13.3 are responsible for benign familial neonatal convulsions (BFNC), a rare monogenic idiopathic epilepsy. Here we report the determination of the detailed genomic structure of KCNQ2, and use of this information in mutational analysis. There are at least 18 exons, occupying more than 50 kb of genomic DNA. Several formerly unknown polymorphisms and splice variants as well as a new single base pair deletion mutation of unusual localization are described. In addition to facilitating more effective mutation detection among BFNC patients, the results presented here provide the basis for analysing the role of KCNQ2 in other types of epilepsy. Received: 24 November 1998 / Accepted: 8 January 1999     

Decreased thermodynamic stability as a crucial factor for familial amyloidotic polyneuropathy

A single mutation in the wild-type transthyretin (WT TTR) such as V30M causes a familial amyloidotic polyneuropathy disease. Comparison of the three-dimensional crystal structures of WT and V30M does not tell much about the reason. High-pressure NMR revealed that at neutral pH both WT and V30M exist as equilibrium between the native tetramer and the dissociated/unfolded monomer. The native tetramer is highly stable in WT (deltaG(0)=104 kJ/mol at 37 degrees C, pH 7.1), but the stability is significantly reduced in V30M (deltadeltaG(0)=-18 kJ/mol), increasing the fraction of the unfolded monomer by a 1000-fold. Significant reduction of thermodynamic stability of WT TTR by mutation could be a crucial factor for familial amyloidotic polyneuropathy.     

Benign familial infantile convulsions: mapping of a novel locus on chromosome 2q24 and evidence for genetic heterogeneity

In 1997, a locus for benign familial infantile convulsions (BFIC) was mapped to chromosome 19q. Further data suggested that this locus is not involved in all families with BFIC. In the present report, we studied eight Italian families and mapped a novel BFIC locus within a 0.7-cM interval of chromosome 2q24, between markers D2S399 and D2S2330. A maximum multipoint HLOD score of 6.29 was obtained under the hypothesis of genetic heterogeneity. Furthermore, the clustering of chromosome 2q24-linked families in southern Italy may indicate a recent founder effect. In our series, 40% of the families are linked to neither chromosome 19q or 2q loci, suggesting that at least three loci are involved in BFIC. This finding is consistent with other autosomal dominant idiopathic epilepsies in which different genes were found to be implicated.     

Lamina-associated polypeptide 2-alpha forms homo-trimers via its C terminus, and oligomerization is unaffected by a disease-causing mutation

The nucleoplasmic protein, Lamina-associated polypeptide (LAP) 2alpha, is one of six alternatively spliced products of the LAP2gene, which share a common N-terminal region. In contrast to the other isoforms, which also share most of their C termini, LAP2alpha has a large unique C-terminal region that contains binding sites for chromatin, A-type lamins, and retinoblastoma protein. By immunoprecipitation analyses of LAP2alpha complexes from cells expressing differently tagged LAP2alpha proteins and fragments, we demonstrate that LAP2alpha forms higher order structures containing multiple LAP2alpha molecules in vivo and that complex formation is mediated by the C terminus. Solid phase binding assays using recombinant and in vitro translated LAP2alpha fragments showed direct interactions of LAP2alpha C termini. Cross-linking of LAP2alpha complexes and multiangle light scattering of purified LAP2alpha revealed the existence of stable homo-trimers in vivo and in vitro. Finally, we show that, in contrast to the LAP2alpha-lamin A interaction, its self-association is not affected by a disease-linked single point mutation in the LAP2alpha C terminus.     

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.     

Reactivating PP2A by FTY720 as a novel therapy for AML with C-KIT tyrosine kinase domain mutation

The tyrosine kinase domain (TKD) mutations of receptor tyrosine kinase C-KIT are associated with a poor prognosis in acute myeloid leukemia (AML). However, the underlying mechanisms are not fully understood. We found the activity of protein phosphatase 2A (PP2A), a human tumor suppressor whose dysfunction contributes to malignant cell behavior, was significantly decreased in AML subgroups harboring C-KIT/D816V and AML cell line Kasumi-1 bearing C-KIT/N822K mutation. Primary AML cells and various AML cell lines were treated with PP2A activator FTY720. FTY720 showed a toxic effect in all leukemic cells, especially for cells harboring C-KIT/TKD mutation. Furthermore, FTY720-induced toxicity in AML leukemic cells was mediated by restoration of PP2A activity, via down-regulation of PP2A inhibitor SET, dephosporylation of PP2A-C(TYR307), and up-regulation of relevant PP2A subunit A and B55α. Our research indicates that the decreased PP2A activity in AML harboring C-KIT/TKD mutation may make the restoration of PP2A activity a novel therapy for AML patients with C-KIT/TKD mutation.     

Hypoosmotic cell swelling as a novel mechanism for modulation of cloned HCN2 channels

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.     

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.     

The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation

The tumor suppressor phosphatase PTEN regulates cell migration, growth, and survival by dephosphorylating phosphatidylinositol second messengers and signaling phosphoproteins. PTEN possesses a C-terminal noncatalytic regulatory domain that contains multiple putative phosphorylation sites, which could play an important role in the control of its biological activity. The protein kinase CK2 phosphorylated, in a constitutive manner, a cluster of Ser/Thr residues located at the PTEN C terminus. PTEN-phosphorylated defective mutants showed decreased stability in comparison with wild type PTEN and were more rapidly degraded by the proteasome. Inhibition of PTEN phosphorylation by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole also diminished the PTEN protein content. Our results support the notion that proper phosphorylation of PTEN by CK2 is important for PTEN protein stability to proteasome-mediated degradation.     

Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.     

A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis

The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.     

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.     

Open-channel block by the cytoplasmic tail of sodium channel beta4 as a mechanism for resurgent sodium current

Voltage-gated sodium channels with "resurgent" kinetics are specialized for high-frequency firing. The alpha subunits interact with a blocking protein that binds open channels upon depolarization and unbinds upon repolarization, producing resurgent sodium current. By limiting classical inactivation, the cycle of block and unblock shortens refractory periods. To characterize the blocker in Purkinje neurons, we briefly exposed inside-out patches to substrate-specific proteases. Trypsin and chymotrypsin each removed resurgent current, consistent with established roles for positively charged and hydrophobic/aromatic groups in blocking sodium channels. In Purkinje cells, the only known sodium channel-associated subunit that has a cytoplasmic sequence with several positive charges and clustered hydrophobic/aromatic residues is beta4 (KKLITFILKKTREK; beta4(154-167)). After enzymatic removal of block, beta4(154-167) fully reconstituted resurgent current, whereas scrambled or point-mutated peptides were ineffective. In CA3 pyramidal neurons, which lack beta4 and endogenous block, beta4(154-167) generated resurgent current. Thus, beta4 may be the endogenous open-channel blocker responsible for resurgent kinetics.     

Formation of a fairly stable diazoate intermediate of 5-methyl-2'-deoxycytidine by HNO2 and NO, and its implication to a novel mutation mechanism in CpG site

The intermediate produced from 5-methyl-2'-deoxycytidine ((5me)dCyd) by HNO2 and NO treatments was isolated and characterized. When 10mM (5me)dCyd was incubated with 100mM NaNO2 at pH 3.7 and 37 degrees C, a previously unidentified product was formed. The product was identified as a diazoate derivative of (5me)dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate ((5me)dCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 microM (0.023% yield). When an aqueous solution of 10mM (5me)dCyd (10 mL) was bubbled by NO at 37 degrees C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 micromol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37 degrees C), the diazoate was converted to dThd exclusively with a first order rate constant k=9.1x10(-6) x s(-1) (t(1/2)=21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of (5me)dCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO.     

Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.     

Counter current transfer of PGF2 alpha in the mesometrial vessels as a mechanism for prevention of luteal regression in early pregnancy

A new concept has been presented on the mechanism protecting the corpus luteum during oestrous cycle, early pregnancy and pseudopregnancy induced by oestrogens. The concept is based on the recently discovered mechanism of back transfer of prostaglandin F2 alpha from the broad ligament vasculature into the uterus and on the participation of oestrogen in this process. The morphological facilitates for counter-current transfer of PGF2 alpha in the area of mesometrial vasculature and ability the uterus to bind PGF2 alpha were presented. It has been concluded that the process of PGF2 alpha back-transfer from mesometrial vasculature into the uterus may reduce in uterine venous blood the amplitude of PGF2 alpha pulses and by this way may reduce the penetration of prostaglandin into subovarian area and from there to the corpus luteum.