Cis Ca2 Research Articles

Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

Calcium regulation of Ca2+-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca2+ permeability. The molecular mechanisms associated with PC2 regulation by Ca2+ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2iv), functionally responds to changes in intracellular (cis) Ca2+. In this study we determined the regulatory effect(s) of Ca2+-sensitive and -insensitive actin-binding proteins (ABPs) on PC2iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2iv channel function in the presence of cis Ca2+, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2iv channel function in the presence, but no effect in the absence of cis Ca2+. Gelsolin stimulated PC2iv channel function in the presence, but not the absence of cis Ca2+. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2iv channel function both in the presence and absence of Ca2+. The distinct effect(s) of the ABPs on PC2iv channel function demonstrate that Ca2+ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca2+-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation.

Calcium Channel Dysfunction in a Mutant Mouse Model of Malignant Hyperthermia(CaV1.1 R174W)

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that is triggered by exposure to volatile anesthetics. MH has been studied extensively in mice and pigs carrying causative mutations in the type 1 ryanodine receptor (RyR1). However, no in vivo information exists regarding how mutations in the skeletal muscle L-type Ca2+ channel (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line carrying the R174W mutation. Homozygous R174W mice ambulated efficiently, reproduced and had normal lifespans. When exposed to isoflurane, homozygous R174W mice entered a hypermetabolic state ending ultimately in death. On the ultrastructural level, R174W muscle displayed limited and variable changes: some variability of the SR calsequestrin content, displacement of mitochondria in some soleus fibers of aged mice and occasional accumulation of SR stacks. On the cellular level, homozygous R174W muscle had elevated resting myoplasmic Ca2+ levels that were greatly increased upon exposure to isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous R174W mice lacked L-type Ca2+ current even though intramembrane charge movements of were of similar magnitude and voltage-dependence to those recorded from wild-type fibers. Ca2+ released from the SR in response to depolarization was substantially reduced in homozygous R174W fibers suggesting a depleted SR Ca2+ store. Lipid bilayer recordings showed that the Po of RyR1s isolated from homozygous R174W mice was significantly increased at all cis Ca2+ concentrations (Feng et al., this meeting). Taken together, our results support a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2+ leak without affecting EC coupling per se. This work was supported by grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend from 2T32AG000279-11.

Calcium Transport and Local Pool Regulate Polycystin-2 (TRPP2) Function in Human Syncytiotrophoblast

Polycystin-2 (PC2, TRPP2) is a Ca2+-permeable, nonselective cation channel implicated in Ca2+ transport and epithelial cell signaling. Although PC2 may contribute to Ca2+ transport in human term placenta, the regulatory mechanisms associated with Ca2+ handling in this tissue are largely unknown. In this work we assessed the regulation by Ca2+ of PC2 channel function from a preparation of apical membranes of human syncytiotrophoblast (PC2hst) reconstituted in a lipid bilayer system. Addition of either EGTA or BAPTA to the cis hemi-chamber, representing the cytoplasmic domain of the channel, and lowering Ca2+ to ∼0.6–0.8 nM, inhibited spontaneous PC2hst channel activity, with a time response dependent on the chelator tested. EGTA reduced PC2hst channel currents by 86%, with a t1/2 = 3.6 min, whereas BAPTA rapidly and completely (100%) eliminated channel activity with a t1/2 = 0.8 min. Subsequent titration with Ca2+ reversed the inhibition, which followed a Hill-type function with apparent dissociation constants of 1–5 nM, and 4 Ca2+ binding sites. The degree of inhibition by the cis Ca2+ chelator largely depended on increasing trans Ca2+. This was consistent with measurable Ca2+ transport through the channel, feeding the regulatory sites in the cytoplasmic domain. Interestingly, the reconstituted in vitro translated PC2 (PC2iv) was completely insensitive to Ca2+ regulation, suggesting that the regulatory sites are not intrinsic to the channel protein. Our findings demonstrate the presence of a Ca2+ microdomain largely accessible through the channel that controls PC2 function in human syncytiotrophoblast of term placenta.

Property of large conductance Ca2+-activated K+ channels from Fasciola hepatica incorporated into planar lipid bilayer

Fasciola hepatica causes biliary epithelial hyperplasia and obstructive jaundice in humans and animals. Using a planar lipid bilayer technique, we further characterized the single channel property of large conductance K+-permeable channels that were previously identified from F. hepatica. The single channel conductance was 254.7±17.9pS under a symmetrical 200/200mM (cis/trans) KCl gradient. Open state probability (Po) varied from channel to channel at a given membrane potential and Ca2+ concentration, but increased with voltage (−60 to +40mV) and cis Ca2+ (1–200μM). Under a near bi-ionic condition of 200mM [K+]cis/200mM [Na+]trans, the permeability ratio of K+ to Na+ was 5.0. Charybdotoxin (1μM) inhibited Po, whereas tetraethylammonium reduced the conductance (KD=67.8mM). Taken together, the results show that the single channel properties of the large conductance K+-permeable channels in F. hepatica are similar to those of large conductance Ca2+-activated K+ (BK) channels in general, but distinct from typical BK channels in the extent of voltage- and Ca2+-dependence, as well as permeability to Na+. This study further reveals a variant BK channel in F. hepatica that could serve as a new drug target to treat fascioliasis.

Gating of the Purified Human Cardiac Ryanodine Receptor (hRyR2) in the Absence of Regulatory Accessory Proteins

The cardiac ryanodine receptor (RyR2) mediates Ca2+ efflux from intracellular stores to effect myocyte contraction during excitation-contraction coupling. Mutations in this channel perturb Ca2+ release function, leading to triggered arrhythmias that may cause sudden cardiac death (SCD). The exact molecular mechanisms by which SCD-linked RyR2 dysfunction occurs constitutes a burgeoning area of cardiac research. Most studies so far have concentrated on the secondary effects of mutation on channel function by virtue of affecting channel modification by phosphorylation and accessory protein binding, with no great emphasis on elucidating the gating mechanisms of the channel itself. Our aim is to elucidate the mechanistic basis of wild-type (WT) hRyR2 activation by its primary activating ligand, Ca2+, under precisely controlled conditions in the absence of any accessory proteins with a view to determining the effect of mutation on hRyR2 gating in the same way. hRyR2 channels, recombinantly expressed in HEK293 cells, were purified and studied at the single channel level in symmetrical 210mM KCl under reducing conditions. Trans (luminal) Ca2+ was buffered at 50nM using EGTA, while cis (cytosolic) Ca2+ buffering was stringently controlled using EGTA, HEDTA and NTA to achieve free Ca2+ concentrations in the range of 0-500 μM. Preliminary data obtained from sigmoidal dose-response curves of Po vs pCa for 10 WT hRyR2 channels yields an EC50 of 3.25±1.04 μM, resulting in a maximum Po greater than 0.8 (in 5 out of 10 channels). This increase in Po resulted from an increase in the frequency of channel openings, until Po of 0.8 - above which any increases in Po resulted from an increase in open times. Likely gating mechanisms will be discussed with a view to mutant channel analysis.Supported by the British Heart Foundation

Dust From Pig Confinement Facilities Bind to and Activate Skeletal Muscle Ryanodine Receptor Calcium-Release Channel (RyR1)

Individuals working in industrial pig farms and residents in communities surrounding these confinement facilities report increased muscle weakness and fatigue. However, causative factors and molecular mechanisms responsible for this remain poorly characterized. In this study we investigated whether dust from confinement facilities contains components that can bind to and activate RyR1. Dust collected 1–2 meters from the ground of pig confinement facilities in Nebraska were extracted with chloroform, filtered and rotor evaporated to dryness. The residues were redissolved in hexane:chloroform (20:1) and precipitates herein referred to as HEX-INS were filtered and air-dried. Thin layer chromatography revealed three major compounds in HEX-INS fractions and 1H NMR suggests these compounds contained fatty acid moieties. In binding assays, HEX-INS displaced [3H]ryanodine from rabbit skeletal muscle RyR1 in a dose-dependent manner, with an IC50 of 2.0μg/ml. HEX-INS also displaced [3H]ryanodine from RyR2 (dog heart), but was significantly less potent (IC50 of 21.0μg/ml). HEX-INS displacement curves were parallel to that of ryanodine and occurred principally at activating Ca2+ concentrations. In single channel studies, HEX-INS increased open probability of RyR1 in a dose-dependent manner (3.5 to 14.0μg/ml, cis Ca2+ 3μM). At 17.5μg/ml, HEX-INS induced RyR1 in a state of reduced conductance (55.2% of maximum). This subconductance state was more likely to occur and persist at positive holding potentials. Increasing HEX-INS further to 21.0μg/ml resulted in reversible channel closure. At a higher cis Ca2+ (20μM), lower amounts of HEX-INS were required for induction of the subconductance state and channel closure. These data are the first to demonstrate that pig confinement dust contains component(s) that selectively bind to and activate RyR1, providing a mechanism rationale for reported muscle weakness and fatigue. (Supported in part by grants from NIH to DJR and KRB)

Presence of the nucleic acid channel in renal brush-border membranes: allosteric modulation by extracellular calcium

We have previously described a cell surface channel complex that is highly selective for nucleic acid (6, 7). The channel complex was purified to homogeneity by solubilizing renal brush-border membranes (BBM) with CHAPS and separation by liquid chromatography. It was characterized by reconstitution in planar lipid bilayers. The channel consists of a pore-forming subunit that is blocked by heparan sulfate and a regulatory subunit that is blocked by L-malate (7). The current studies were performed to compare the characteristics of the nucleic acid-conducting channel in native BBM with the characteristics that have been determined for the complex reconstituted from purified proteins. BBM were purified by differential centrifugation and reconstituted in lipid bilayers. Current was not observed until oligodeoxynucleotide (ODN) was added. Conductance was 9.1 +/- 0.9 pS; rectification and voltage dependence were not observed. Reversal potential (E(rev)) shifted to +14 +/- 0.1 mV by a 10-fold gradient for ODN but was not altered when gradients were created for any other ion. Open probability increased significantly with an increase in Ca(2+) on the trans chamber of the bilayer apparatus. Changes in cis Ca(2+) were without effect. Addition of L-malate to the cis chamber or heparan sulfate to the trans chamber significantly reduced the open probability of the channel. These data demonstrate that the nucleic acid channel in BBM is electrophysiologically and pharmacologically identical to that previously reported for purified protein and demonstrate that a nucleic acid-conducting channel is a component of renal BBM.

Thermodynamically Irreversible Gating of Ryanodine Receptors in Situ Revealed by Stereotyped Duration of Release in Ca2+ Sparks

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca2+ release underlying Ca2+ sparks in intact cardiac myocytes exhibits a prominent mode at ∼8ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than ∼35ms and a mode at ∼90ms. These results indicate that, regardless of whether Ca2+ sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4μM) and trans Ca2+ (10mM), suggesting that the irreversibility for Ca2+ spark genesis may reside at a supramolecular level. Modeling suggests that Ca2+-induced Ca2+ release among adjacent RyRs may couple the external energy derived from Ca2+ gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca2+ sparks.

PH and external Ca2+ regulation of a small conductance Cl− channel in kidney distal tubule

A single channel characterization of the Cl− channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl− channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca2+ regulation of a small conductance Cl− channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability PO. Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel PO with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca2+ concentration, with a dose-dependent increase in channel activity as a function of the cis Ca2+ concentration. It is concluded on the basis of these results that the small conductance Cl− channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca2+.

Calcium dependence of C-type natriuretic peptide-formed fast K(+) channel.

The lipid bilayer technique was used to characterize the Ca(2+) dependence of a fast K(+) channel formed by a synthetic 17-amino acid segment [OaCNP-39-(1-17)] of a 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchus anatinus) venom (OaV). The OaCNP-39-(1-17)-formed K(+) channel was reversibly dependent on 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic) Ca(2+) concentration ([Ca(2+)](cis)). The channel was fully active when [Ca(2+)](cis) was >10(-4) M and trans (luminal) Ca(2+) concentration was 1.0 mM, but not at low [Ca(2+)](cis). The open probability of single channels increased from zero at 1 x 10(-6) M cis Ca(2+) to 0.73 +/- 0.17 (n = 22) at 10(-3) M cis Ca(2+). Channel openings to the maximum conductance of 38 pS were rapidly and reversibly activated when [Ca(2+)](cis), but not trans Ca(2+) concentration (n = 5), was increased to >5 x 10(-4) M (n = 14). Channel openings to the submaximal conductance of 10.5 pS were dominant at >/=5 x 10(-4) M Ca(2+). K(+) channels did not open when cis Mg(2+) or Sr(2+) concentrations were increased from zero to 10(-3) M or when [Ca(2+)](cis) was maintained at 10(-6) M (n = 3 and 2). The Hill coefficient and the inhibition constant were 1 and 0.8 x 10(-4) M cis Ca(2+), respectively. This dependence of the channel on high [Ca(2+)](cis) suggests that it may become active under 1) physiological conditions where Ca(2+) levels are high, e.g., during cardiac and skeletal muscle contractions, and 2) pathological conditions that lead to a Ca(2+) overload, e.g., ischemic heart and muscle fatigue. The channel could modify a cascade of physiological functions that are dependent on the Ca(2+)-activated K(+) channels, e.g., vasodilation and salt secretion.

Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser687 and FKBP12

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2>A1>A1S>NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by ∼30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at −40mV, with 10μM A1 or 50nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100nM cis Ca2+. Inhibition by A1 was greatest at +40mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100μM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca 2+ Involves Luminal Ca 2+ Sensing Sites

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca 2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 μM to 2 mM Ca 2+ (free) and 3 mM ATP (total) on the cytosolic ( cis) side and 20 μM to 20 mM Ca 2+ on the luminal ( trans) side of the channel and with Cs + as the charge carrier. Under conditions of low trans Ca 2+ (20 μM), increasing cis Ca 2+ from 0.1 to 10 μM caused a gradual increase in channel open probability ( P o). Elevating cis Ca 2+ above 100 μM resulted in a gradual decrease in P o. Elevating trans [Ca 2+] enhanced channel activity (EC 50 ≈ 2.5 mM at 1 μM cis Ca 2+) primarily by increasing the frequency of channel openings. The dependency of P o on trans [Ca 2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca 2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N, N-tetraacetic acid. Elevated luminal Ca 2+ enhanced the sensitivity of the channel to activating cytosolic Ca 2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca 2+. Potentiation of P o by increased luminal Ca 2+ occurred irrespective of whether the electrochemical gradient for Ca 2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca 2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca 2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca 2+ are mediated by distinct Ca 2+-sensitive site(s) at the luminal face of the channel or associated protein.

Activation of the cardiac ryanodine receptor by sulfhydryl oxidation is modified by Mg2+ and ATP.

The reactive disulfide 4,4'-dithiodipyridine (4,4' DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10(-7) M (in the absence of Mg2+ and ATP), increased within approximately 1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg2+ (10(-7) M cis Ca2+) or by 10 mM cis Mg2+ (10(-3) M cis Ca2+), or activated by 4 mM ATP (10(-7) M cis Ca2+), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity. Po and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.

Interaction of S100A1 with the Ca2+ Release Channel (Ryanodine Receptor) of Skeletal Muscle

In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium.

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1-10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

Inositol polyphosphates modify the kinetics of a small chloride channel in skeletal muscle sarcoplasmic reticulum.

The actions of D-myo-inositol 1,4,5-trisphosphate (IP3) and D-myo-inositol 1,3,4,5-tetrakisphospate (IP4) on small chloride (SCl) channels from rabbit skeletal muscle sarcoplasmic reticulum are reported. We find that the inositol polyphosphates (6-40 microM) are potent reversible blockers of SCl channels in lipid bilayers at -40 mV with >10(-5) m cis (cytoplasmic) Ca2+ when added to the cis, but not trans, chamber. IP3 or IP4 at 20 microM reduced the mean open time from 89 +/- 16 msec to 11 +/- 2 msec or to 8.0 +/- 1.0 msec respectively, by abolishing the longest time constant component in the open time distribution. Neither IP3 nor IP4 altered the six single-channel conductance levels. The fraction of low conductance events increased approximately 4-fold and the dwell time at the lower conductance levels increased approximately 3-fold. Channel gating was altered so that most transitions were between the closed level and an open level, in contrast to control channels which remained open for long periods with many transitions between the six open levels. The actions of the inositol polyphosphates were: (1) not prevented by 20 microg/ml cis heparin (an IP3 receptor blocker); (2) mimicked by 10 microM cis synthetic inositol polyphosphates, L-chiro-inositol 1,4,6-trisphosphate and L-chiro-inositol 1,4,6-trisphosphorothionate (which do not bind to IP3 receptors); (3) mimicked by cis additions of the polyanions heparin or hepran (20 microg/ml each) and vanadate (500 microM). The results suggest that an interaction between polyanions and SCl channels would allow the channels to be modulated in vivo by inositol polyphosphates.

Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12

FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs.

Analytical and Clinical Evaluation of a Procedure for Measuring CA 15-3 in the IMx Analyser

CA 15-3 is the most widely used tumour marker in the follow-up of patients with breast cancer. Its postoperative blood level is closely related to the response to the treatment applied and to the evolution of the disease. In this study, we assessed both the clinical and analytical features of a new microparticle enzyme immunoassay for CA 15-3 quantification in the Abbott IMx system. In the precision study, the coefficients of variation within runs, between runs and between laboratories were 3.5, 5.1 and 6.5% or less, respectively. The analytical sensitivity of the assay was 0.11 U/ml. Linear regression analyses of the IMx CA 15-3 assay with the Centocor and CIS CA 15-3 RIAs gave correlation coefficients of 0.88 and 0.95, respectively. The upper limit of the reference range, obtained from serum of healthy women, was 27.3 U/ml, while the ROC curve analysis for patients with active breast cancer compared to patients with no evidence of the disease gave an optimum cutoff of 33 U/ml (sensitivity 0.76 and specificity 0.87). The overall agreement of the three methods was over 90%.

Description of modal gating of the cardiac calcium release channel in planar lipid membranes

Single channel activity of the cardiac ryanodine-sensitive calcium-release channel in planar lipid membranes was studied in order to elucidate the calcium-dependent mechanism of its steady-state behavior. The single channel kinetics, observed with Cs+ as the charge carrier at different activating (cis) Ca2+ concentrations in the absence of ATP and Mg2+, were similar to earlier reports and were extended by analysis of channel modal behavior. The channel displayed three episodic levels of open probability defining three gating modes: H (high activity), L (low activity), and I (no activity). The large difference in open probabilities between the two active modes resulted from different bursting patterns and different proportions of two distinct channel open states. I-mode was without openings and can be regarded as the inactivated mode of the channel; L-mode was composed of short and sparse openings; and H-mode openings were longer and grouped into bursts. Modal gating may explain calcium-release channel adaptation (as transient prevalence of H-mode after Ca2+ binding) and the inhibitory effects of drugs (as stabilization of mode I), and it provides a basis for understanding the regulation of calcium release.

IP 3 receptor purified from liver plasma membrane is an (1,4,5)IP 3 activated and (1,3,4,5)IP 4 inhibited calcium permeable ion channel

The IP 3 receptor is involved in Ca 2+ mobilization from intracellular stores. Recently, we purified an inositol (1,4,5)-trisphosphate receptor from rat liver plasma membrane (LPM-IP 3R) [Schäfer R. Hell K. Fleischer S. (1993) Purification of an IP 3 receptor from liver plasma membrane. Biophys. J. 66, A146] . The purified LPM-IP 3 receptor was incorporated into vesicle derived planar bilayers and its channel properties characterized. The receptor displayed ion channel activity that was activated by inositol (1,4,5-trisphosphate [(1,4,5)IP 3] (1 μM) and inhibited by inositol (1,3,4,5)-tetrakisphosphate (IC 50 ∼1 μM) and by heparin (IC 50 ∼20 gm/ml). The channel displays a unitary conductance of 9 pS, and 13 pS in symmetrical 100 mM and 500 mM KCI, respectively, and in symmetrical 250 mM cesium methanesulfonate the slope conductance is 11 pS. Activation by (1,4,5)IP 3 is specific to the cis-side of the chamber, equivalent to the cytoplasmic face. The receptor is a Ca 2+ permeable ion channel based on ion selectivity (Ca 2+ > K + > Na + ⪢ Cl −). The LPM-IP 3 receptor was also permeable to Cs (Cs + ≥ K +), similar to other intracellular Ca 2+ release channels, i.e. the IP 3 receptor from brain and smooth muscle (IP 3R-1) and the ryanodine receptor from skeletal muscle (RyR-1) and heart (RyR-2). Channel activity is not voltage dependent (± 100 mV applied voltage). The channel is activated by ATP and Ca 2+. The open probability of the (1,4,5)IP 3 activated channel activity displays a bell shaped response to cis Ca 2+ ion concentration of our system. The LPM-IP 3 receptor differs from intracellular IP 3R-1 in that the Ca 2+ and ATP concentration required for maximum activation is about 10 times higher as compared with IP 3R-1 from brain cerebellum and smooth muscle. We conclude that the LPM-IP 3 receptor is an (1,4,5)IP 3 activated Ca 2+ permeable ion channel. The implication of our studies is that in liver, (1,4,5)IP 3 regulates Ca 2+ influx via the plasma membrane.