Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx.

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 µM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Observations of calcium dynamics in cortical secretory vesicles.

Calcium (Ca(2+)) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca(2+) concentration in the secretory vesicles was estimated to be in the range of ∼10 to 100 µM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca(2+) dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca(2+) signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Ca(v)2.1) Ca(2+) channels. Blocking the p-type Ca(2+) channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca(2+) dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca(2+) indicator fluorescence. The results suggest that the free Ca(2+) content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process.

Cell swelling-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca2+ and is tetanus toxin resistant.

UNLABELLED: Cell swelling-induced insulin secretion represents an alternative pathway of stimulation of insulin secretion. INS-1E rat tumor beta cells do not release insulin in response to cell swelling in presence of Ca(2+) despite a good response to glucose challenge and appropriate increase in cell volume. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity. Objective of this study was further characterization of the role of Ca(2+) in secretory process in INS-1 and INS-1E cell lines. Ca(2+) depleted hypotonic medium with 10 muM BAPTA/AM (intracellular chelator) induced insulin secretion from both types of cells. We demonstrated expression of L-type Ca(2+) channel Ca(v)1.2 and non-L-type Ca(2+) channels Ca(v)2.1 (P/Q-type), Ca(v)2.2 (N-type), and Ca(v)3.1 (T-type) in both cell lines. Inhibition of L type channel with nifedipine and/or P/Q type with omega-agatoxin IVA enabled distinct response to hypotonic medium also in INS-1E cells. Tetanus toxin (TeTx) in medium containing Ca(2+) and a group of calcium channel blockers inhibited hypotonicity-induced insulin secretion from INS-1 cells but not from INS-1E cells. CONCLUSION: Hypotonicity-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca(2+), does not require intracellular Ca(2+) and is TeTx resistant.

Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.

P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca(V)2.1 alpha1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca(V)2 channels and suggest that the ability of individual Ca2+ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.

The ataxic groggy rat has a missense mutation in the P/Q-type voltage-gated Ca2+ channel alpha1A subunit gene and exhibits absence seizures.

The groggy rat (strain name; GRY) exhibits ataxia, an unstable gait, and paroxysmal severe extension of the entire body. Adults show a reduction in size of the cerebellum and presynaptic and axon terminal abnormalities of Purkinje cells. These neurological abnormalities are inherited in an autosomal recessive manner, and the causative mutation has been named groggy (gry). In this study, we mapped gry on rat chromosome 19 and found a nonconservative missense (M251K) mutation in the alpha(1A) subunit of the P/Q-type voltage-gated Ca(2+) channel gene (Cacna1a) within the gry-critical region. This mutation was located at a highly conserved site close to the ion-selective pore and led to the shortening of the inactivation phase of the Ca(2+) channel current without a change of peak current density or current-voltage relationship in whole cell patch recordings of the recombinant Ca(2+) channel expressed in HEK cells. It has been well established that mice with a mutation at Cacna1a such as tottering and leaner show absence seizures. The Cacna1a-mutant GRY rat also exhibited absence-like seizures from 6 to 8 weeks of age, which were characterized by bilateral and synchronous 7-8 Hz spike-and-wave discharges concomitant with sudden immobility and staring, on cortical and hippocampal EEGs. The pharmacological profile of the seizures was similar to that of human absence epilepsy: the seizures were inhibited by ethosuximide and valproic acid but not phenytoin. Thus, the GRY rat with P/Q-type Ca(2+) channel disorders is a useful model for studying absence epilepsy and Cacna1a-related diseases.

Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases.

Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozen-hydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na(+)/K(+)-ATPase, rabbit skeletal Ca(2+)-ATPase and scallop Ca(2+)-ATPase in order to evaluate the potential for sub-nanometer resolution as well as the robustness in the presence of disorder. We found that Fourier-Bessel methods perform better for well-ordered samples of skeletal Ca(2+)-ATPase and Na(+)/K(+)-ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca(2+)-ATPase, which was too disordered to analyze by Fourier-Bessel methods.

Lambert-Eaton myasthenic syndrome as an autoimmune calcium channelopathy.

Lambert-Eaton myasthenic syndrome, often associated with small-cell lung carcinoma, is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC)(P/Q-type) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. The motor nerve terminal and carcinoma cell may share a common antigen. The study using synthetic peptides and recombinant protein specified the extracellular S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Also, the study by use of peptides and recombinant protein corresponding to synaptotagmin I suggested that in this functionally VGCC-associated presynaptic protein, the segment which exposes extracellularly during exocytosis can be immunogenic for the syndrome.

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.

Why we must move on from the E1E2 model for the reaction cycle of the P-type ATPases.

Recent progress regarding the structure of the Ca(2+)-translocating ATPase of sarcoplasmic reticulum in several conformational states, and a substantial accumulation of biochemical information about this and other P-type ATPases, have put everything in place for the final convergence of biochemistry and structure that will lead to a complete understanding of the molecular mechanism of these membrane transport enzymes. But the common paradigm used to describe the reaction cycle of the P-type ATPases, the E1E2 model, is seriously flawed, and this is hindering our progress toward this goal. In this paper, it is first shown why the E1E2 model must be discarded. This is followed by a description of the P-type ATPase catalytic cycle that is much more consistent with the structural and biochemical information now available for these enzymes, and also brings to light the origin of the forces that drive the key reaction in the active transport cycle where high-affinity ion-binding sites are converted to low-affinity binding sites capable of releasing the transported ions against a considerable concentration gradient. This new model will therefore serve us better as we seek to unravel the final details of the molecular mechanism of active ion transport catalyzed by these enzymes. It is thus time to move on from the traditional E1E2 model.

Long-term regulation of voltage-gated Ca(2+) channels by gabapentin.

Gabapentin (GBP) is a gamma-aminobutyric acid analog effective in the treatment of seizures. A high-affinity interaction between GBP and the alpha(2)delta subunit of the voltage-gated Ca(2+) channels has been documented. In this report, we examined the effects of the chronic treatment with GBP on neuronal recombinant P/Q-type Ca(2+) channels expressed in Xenopus oocytes. GBP did not affect significantly the amplitude or the voltage dependence of the currents. Exposure to the drug did, however, slow down the kinetics of inactivation in a dose-dependent fashion. In addition, biochemical analysis showed that the integrity of Ca(2+) channel complex is not apparently affected by GBP binding, suggesting that chronic treatment with the drug might cause the channel kinetic modification through subtle conformational changes of the protein complex.

Role of Thr(11) in the binding of omega-conotoxin MVIIC to N-type Ca2+ channels.

As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.Gt;Asp, Phe, Tyr for P/Q-type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N-type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N-type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.

The spider toxin omega-Aga IIIA defines a high affinity site on neuronal high voltage-activated calcium channels.

The spider toxin omega-agatoxin IIIA (omega-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of omega-Aga-IIIA to a single population of high affinity sites, with K(d) = approximately 9 pm and B(max) = approximately 1.4 pmol/mg protein. Partial inhibition of omega-Aga-IIIA binding by omega-conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. omega-Aga-IIIA binds to recombinant alpha(1B) and alpha(1E) calcium channels with a similar high affinity (K(d) = approximately 5-9 pm) in apparent one-to-one fashion. Results from recombinant alpha(1B) binding experiments demonstrate virtually identical B(max) values for omega-Aga-IIIA and omega-conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that omega-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.

Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells.

The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini.

Calcium pumps: structural basis for and mechanism of calcium transmembrane transport.

Eukaryotic cells remove calcium from the cytosol using P-type pumps in the plasma membrane and in the sarco(endo)plasmic reticulum. These pumps share membrane topography and general mechanism of action, but differ in regulatory properties. Recent advances in the field include the three-dimensional structure of the sarco(endo)plasmic reticulum and further understanding of the transcriptional regulation of the plasma membrane P-type pump by calcium.

Spinocerebellar ataxia type 6 mutation alters P-type calcium channel function.

Abnormal CAG repeat expansion in the alpha1A voltage-dependent calcium channel gene is associated with spinocerebellar ataxia type 6, an autosomal dominant cerebellar ataxia with a predominant loss of the Purkinje cell. A reverse transcriptase-polymerase chain reaction analysis of mRNA from mouse Purkinje cells revealed a predominant expression of the alpha1A channel lacking an asparagine-proline (NP) stretch in the domain IV (alpha1A(-NP)). Human alpha1A channels carrying various polyglutamine length with or without NP were expressed in HEK293 cells, and channel properties were compared using a whole-cell voltage clamp technique. alpha1A(-NP), corresponding to P-type channel, with 24 and 28 polyglutamines found in patients showed the voltage dependence of inactivation shifting negatively by 6 and 11 mV, respectively, from the 13 polyglutamine control. Contrarily, the alpha1A channel with NP (alpha1A(+NP)), corresponding to Q-type channel, with 28 polyglutamines exhibited a positive shift of 5 mV. These results suggest that altered function of alpha1A(-NP) may contribute to degeneration of Purkinje cells, which express predominantly alpha1A(-NP), due to the reduced Ca(2+) influx resulting from the negative shift of voltage-dependent inactivation. On the other hand, other types of neurons, expressing both alpha1A(-NP) and alpha1A(+NP), may survive because the positive shift of voltage-dependent inactivation of alpha1A(+NP) compensates Ca(2+) influx.

Direct alteration of the P/Q-type Ca2+ channel property by polyglutamine expansion in spinocerebellar ataxia 6.

Spinocerebellar ataxia 6 (SCA6) is caused by expansion of a polyglutamine stretch, encoded by a CAG trinucleotide repeat, in the human P/Q-type Ca(2+) channel alpha(1A) subunit. Although SCA6 shares common features with other neurodegenerative glutamine repeat disorders, the polyglutamine repeats in SCA6 are exceptionally small, ranging from 21 to 33. Because this size is too small to form insoluble aggregates that have been blamed for the cause of neurodegeneration, SCA6 is the disorder suitable for exploring the pathogenic mechanisms other than aggregate formation, whose universal role has been questioned. To characterize the pathogenic process of SCA6, we studied the effects of polyglutamine expansion on channel properties by analyzing currents flowing through the P/Q-type Ca(2+) channels with an expanded stretch of 24, 30, or 40 polyglutamines, recombinantly expressed in baby hamster kidney cells. Whereas the Ca(2+) channels with