Wolfram syndrome-associated mutations lead to instability and proteasomal degradation of wolframin

Wolfram syndrome is caused by mutations in WFS1 encoding wolframin, a polytopic membrane protein of the endoplasmic reticulum. Here, we investigated the molecular pathomechanisms of four missense and two truncating mutations in WFS1. Expression in COS-7 cells as well as direct analysis of patient cells revealed that WFS1 mutations lead to drastically reduced steady-state levels of wolframin. All mutations resulted in highly unstable proteins which were delivered to proteasomal degradation. No wolframin aggregates were found in patient cells suggesting that Wolfram syndrome is not a disease of protein aggregation. Rather, WFS1 mutations cause loss-of-function by cellular depletion of wolframin.     

The WFS1 gene,responsible for low frequency sensorineural hearing loss and Wolfram syndrome,is expressed in a variety of inner ear cells

Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.     

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants.

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ ²-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.     

A long-lasting potentiation of calmodulin-mediated chloride channel activity without a mediation of protein kinase C in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes injected with rat brain poly(A)+RNA were voltage-clamped in a recording solution containing Ca2+, a depolarization pulse induced a transient current, ICl(Ca), which reflects calmodulin-mediated opening of endogenous Cl- channels in response to a Ca2+ influx through Ca2+ channels of brain origin. ICl(Ca) could be repetitively observed with a steady amplitude over 1 h, whereas the response was greatly potentiated for more than 30 min after a brief stimulation of muscarinic or other Ca2(+)-mobilizing receptors. The enhancement of ICl(Ca) was mimicked by an injection of inositol-1,4,5-trisphosphate or by a treatment with A23187, but not affected by treatments that stimulate or inhibit protein kinase C activity. Isolated Ba2+ current flowing through voltage-sensitive Ca2+ channels was not augmented during the facilitation of ICl(Ca). These observations indicate that the endogenous calmodulin/Cl- channel system may memorize an over-threshold increase in the intracellular Ca2+ concentration and potentiate the Ca2(+)-sensitiveness of the Cl- channel. A long-lasting autoregulation of Ca2(+)-dependent ion channel activity is suggested.     

Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-kappaB inhibitors in Crohn's colitis model

We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in alpha-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-kappaB inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-kappaB-dependent pathways.     

Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+β1 subunit of BK(Ca) channel, hSloα+β1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+β1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+β1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+β1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.     

Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K+ channel KAT1 alters current parameters in Xenopus laevis oocytes

Increased guard cell cytosolic [Ca2+] is known to be involved in signal transduction pathways leading to stomatal closure, and inhibit the inward rectifying guard cell K+ channel KAT1. Guard cell calcium-dependent protein kinase (CDPK) has been shown to phosphorylate KAT1; such phosphorylation is known to modulate other K+ channels involved in signal transduction cascades. The work reported here focused on demonstrating CDPK-dependent inhibition of KAT1 currents. A cDNA encoding soybean CDPK was generated and it's translation product was shown to be functional; demonstrating Ca2+-dependent autophosphorylation and phosphorylation of a target protein. Ion currents were monitored using voltage clamp techniques upon expression of KAT1 in Xenopus laevis oocytes. Coexpression of recombinant CDPK with KAT1 in oocytes altered the kinetics and magnitude of induced K+ currents; at a given hyperpolarizing command voltage, the magnitude of KAT1 currents was reduced and the half-time for channel activation was increased. This finding supports a model of Ca2+-dependent ABA inhibition of inward K+ currents in guard cells as being mediated by CDPK phosphorylation of KAT1.     

Gender differences in the modulation of cardiac gene expression by dietary conjugated linoleic acid isomers

In an earlier study, we showed that dietary conjugated linoleic acid (CLA) isomers can exert differential effects on heart function in male and female rats, but the underlying mechanisms for these actions are not known. Cardiomyocyte Ca2+ cycling is a key event in normal cardiac contractile function and defects in Ca2+ cycling are associated with cardiac dysfunction and heart disease. We therefore hypothesized that abnormalities in the sarcolemmal (SL) and sarcoplasmic reticulum (SR)-mediated regulation of intracellular Ca2+ contribute to altered cardiac contractile function of male and female rats owing to dietary CLA isomers. Healthy male and female Sprague-Dawley rats were fed different CLA isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. We determined the mRNA levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a, ryanodine receptor, phospholamban, calsequestrin, Na+-Ca2+-exchanger (NCX), and L-type Ca2+ channel in the left ventricle (LV) by RT-PCR. The SR function was assessed by measurement of Ca2+-uptake and -release. Significant gender differences were seen in the LV NCX, L-type Ca2+ channel, and ryanodine receptor mRNA expression levels in control male and female rats. Dietary CLA isomers in the various forms induced changes in the mRNA levels of SERCA 2a, NCX, and L-type Ca2+ channel in the LV of both male and female hearts. Whereas protein contents of the Ca2+ cycling proteins were altered, changes in SR Ca2+-uptake and -release were also detected in both male and female rats in response to dietary CLA. The results of this study demonstrate that long-term dietary supplementation can modulate cardiac gene expression and SR function in a gender-related manner and may, in part, contribute to altered cardiac contractility.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in store-operated Ca2+ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3' and 5' rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-l polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca2+ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP3F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC50 = 0.5 microM), indicating that InsP3F and LPA principally activate store-operated Ca2+ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP3F-, LPA- or thapsigargin-stimulated Ca2+ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP3F-stimulated Ca2+ inflow and only a very small enhancement of LPA-stimulated Ca2+ in-flow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca2+ inflow through the previously-characterised Ca2+ -specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.     

Regulation of Ca-sensitive inactivation of a 1-type Ca2+ channel by specific domains of beta subunits.

Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.     

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.