scholarly journals Regulation of Type 1 Inositol 1,4,5-Trisphosphate–gated Calcium Channels by InsP3 and Calcium

1999 ◽  
Vol 113 (6) ◽  
pp. 837-849 ◽  
Author(s):  
I.I. Moraru ◽  
E.J. Kaftan ◽  
B.E. Ehrlich ◽  
J. Watras
Keyword(s):  
Mathematical Model ◽  
Ca Channel ◽  
Channel Kinetics ◽  
Channel Activity ◽  
Open Probability ◽  
Intact Cells ◽  
Data Channel ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)–gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443–446; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561–17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding ∼3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.

scholarly journals Inositol 1,4,5-Trisphosphate (InsP3) and Calcium Interact to Increase the Dynamic Range of InsP3 Receptor-dependent Calcium Signaling

1997 ◽  
Vol 110 (5) ◽  
pp. 529-538 ◽  
Author(s):  
Edward J. Kaftan ◽  
Barbara E. Ehrlich ◽  
James Watras
Keyword(s):  
Binding Sites ◽  
Dynamic Range ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Activity ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Low Levels ◽  
Bell Shaped Curve

The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443–446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642–29649; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591–598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 μM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 μM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with Kd < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61–67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192–195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468–470) and a low affinity InsP3 binding site (Kd = 10 μM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.

scholarly journals Calpain-cleaved Type 1 Inositol 1,4,5-Trisphosphate Receptor (InsP3R1) Has InsP3-independent Gating and Disrupts Intracellular Ca2+ Homeostasis

2011 ◽  
Vol 286 (41) ◽  
pp. 35998-36010 ◽  
Author(s):  
Catherine M. Kopil ◽  
Horia Vais ◽  
King-Ho Cheung ◽  
Adam P. Siebert ◽  
Don-On Daniel Mak ◽  
...  
Keyword(s):  
Single Channel ◽  
Open Probability ◽  
Release Channel ◽  
Terminal Fragment ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Functional Consequences ◽  
Carboxyl Terminal ◽  
Trisphosphate Receptor

The type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) is a ubiquitous intracellular Ca2+ release channel that is vital to intracellular Ca2+ signaling. InsP3R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca2+ homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP3R1 and utilized a recombinant truncated form of the channel (capn-InsP3R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP3R1 revealed InsP3-independent gating and high open probability (Po) under optimal cytoplasmic Ca2+ concentration ([Ca2+]i) conditions. However, some [Ca2+]i regulation of the cleaved channel remained, with a lower Po in suboptimal and inhibitory [Ca2+]i. Expression of capn-InsP3R1 in N2a cells reduced the Ca2+ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca2+ loading compared with control cells expressing full-length InsP3R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP3R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP3R1 generates a dysregulated channel that disrupts cellular Ca2+ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP3R1 in a clinically relevant injury model, suggesting that Ca2+ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials.

1988 ◽  
Vol 92 (1) ◽  
pp. 27-54 ◽  
Author(s):  
R L Rosenberg ◽  
P Hess ◽  
R W Tsien
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Detailed Comparison ◽  
Membrane Potentials ◽  
Bay K 8644 ◽  
Ca Channel ◽  
Ca Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Free System

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.

scholarly journals Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

1997 ◽  
Vol 110 (5) ◽  
pp. 503-513 ◽  
Author(s):  
Klaus Schuhmann ◽  
Christoph Romanin ◽  
Werner Baumgartner ◽  
Klaus Groschner
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphatase ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Open Probability ◽  
Intact Cells ◽  
Protein Phosphatase 2B ◽  
Dependent Inhibition ◽  
Inside Out ◽  
Ca2 Channels

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

scholarly journals Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor

2004 ◽  
Vol 279 (44) ◽  
pp. 46242-46252 ◽  
Author(s):  
Larry E. Wagner ◽  
Wen-Hong Li ◽  
Suresh K. Joseph ◽  
David I. Yule
Keyword(s):  
Splice Variants ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Igm Antibody ◽  
Temporal Properties ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Spatio Temporal ◽  
In Cells

Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+signals. In this study, regulation of Ca2+release by phosphorylation of type 1 InsP3R (InsP3R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1, InsP3-induced Ca2+release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca2+release through the S2- S1589E/S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca2+release. The sensitivity of Ca2+release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP3R-1 resulted in robust Ca2+oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.

Abstract 174: Biophysical Basis of Protein Kinase C Inhibition of the Novel alpha1D Calcium Channel

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Mohamed Chahine ◽  
Yongxia Qu ◽  
Mohamed Boutjdir
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Channel ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Kinase C ◽  
Pkc Activation

The recently reported α 1D calcium channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single channel level is not known. Therefore, the effect of PKC activation was studied on α 1D calcium channel expressed in tsA201 cells using cell-attached method. Unitary currents were recorded in the presence of 70 mM Ba 2+ as the charge carrier. Unitary currents were evoked by 500 ms depolarizing pulses from a holding potential of −80 mV every 0.5 Hz. Under basal condition, channel activity was rare and infrequent, however Bay K 8644 (1 μM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by PMA (10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the α 1D calcium channel. This is consistent with a decreased entry of α 1D Ca channel into open states in the presence of PMA. These data show, for the fist time, 1) the α 1D calcium channel activity at the single channel level and 2) the biophysical basis of by which PKC activation inhibits the α 1D calcium channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the α 1D Ca-channel activity and the reduction in whole-cell α 1D Ca current previously reported. Altogether, these data are relevant to the understanding of the patho-physiology of α 1D calcium channel and its regulation by the autonomics.

Identification of a Basolateral Membrane Potassium Channel from Teleost Intestinal Epithelial Cells

1991 ◽  
Vol 159 (1) ◽  
pp. 45-64
Author(s):  
CHRISTOPHER A. LORETZ ◽  
CHARLES R. FOURTNER
Keyword(s):  
Membrane Potential ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
K Channel ◽  
Ca Channel ◽  
Intestinal Epithelial ◽  
Channel Activity ◽  
Open Probability ◽  
Physiological Range

Using patch-clamp techniques, a Ca2+-dependent, voltage-gated K+ channel [K(Ca) channel] was isolated from the basolateral membrane of NaCl-absorbing intestinal epithelial cells of the goby Gillichthys mirabilis. This K(Ca) channel had a high conductance (approximately 150 pS) in the physiological range of membrane potential. Conclusive identification as a K+ channel is supported by dependence of the reversal potential for single-channel current on the K+ concentration gradient and the ability of Ba2+, Cs+ and other pharmacological agents to block the channel. The channel was highly selective for K+ over Na+ (PNa/PK=0.04). Channel activity, expressed as open probability (Po), was dependent on membrane potential with depolarization increasing Po over the physiological range in the presence of Ca2+. Channel activity was also dependent on cytoplasmic-side Ca2+. Po was reduced to near-zero levels following EGTA chelation of Ca2+ in the solution bathing the cytoplasmic face of excised membrane patches; channel activity was most sensitive to changes in Ca2+ concentration between 10nmoll−1 and 10μmoll−1. This K(Ca) channel may be one of several avenues for K+ exit across the basolateral cell membrane and, as such, may play roles in both transepithelial salt transport and maintenance of intracellular ionic composition.

scholarly journals Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

2008 ◽  
Vol 295 (5) ◽  
pp. C1376-C1384 ◽  
Author(s):  
Guiling Zhao ◽  
Adebowale Adebiyi ◽  
Eva Blaskova ◽  
Qi Xi ◽  
Jonathan H. Jaggar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Purinergic Receptor ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Physiological Functions ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

Inositol 1,4,5-trisphosphate receptors (IP3Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP3R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP3R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP3R blockers, reduced Ca2+ wave activation and global intracellular Ca2+ ([Ca2+]i) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP3R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP3R1 being the most abundant isoform at 82% of total IP3R message. IP3R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca2+ wave frequency and global [Ca2+]i but abolished UTP-induced Ca2+ wave activation and reduced the UTP-induced global [Ca2+]i elevation by ∼61%. Antibodies targeting IP3R1 and IP3R1 knockdown reduced UTP-induced nonselective cation current ( Icat) activation. IP3R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca2+ by ∼45%. These data indicate that IP3R1 is the predominant IP3R isoform expressed in rat cerebral artery smooth muscle cells. IP3R1 stimulation contributes to UTP-induced Icat activation, Ca2+ wave generation, global [Ca2+]i elevation, and vasoconstriction. In addition, IP3R1 activation constricts cerebral arteries in the absence of SR Ca2+ release by stimulating plasma membrane Icat.

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