Unitary Ca2+ current through recombinant type 3 InsP3 receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca2+ into the cytoplasm upon binding InsP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open InsP3R channel determines the amount of Ca2+ released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca2+ signals generated in response to extracellular stimuli. Despite its significance, iCa for InsP3R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of iCa through an InsP3R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP3R channels. Within physiological ranges of free Ca2+ concentrations in the ER lumen ([Ca2+]ER), free cytoplasmic [Ca2+] ([Ca2+]i), and symmetric free [Mg2+] ([Mg2+]f), the iCa–[Ca2+]ER relation was linear, with no detectable dependence on [Mg2+]f. iCa was 0.15 ± 0.01 pA for a filled ER store with 500 µM [Ca2+]ER. The iCa–[Ca2+]ER relation suggests that Ca2+ released by an InsP3R channel raises [Ca2+]i near the open channel to ∼13–70 µM, depending on [Ca2+]ER. These measurements have implications for the activities of nearby InsP3-liganded InsP3R channels, and they confirm that Ca2+ released by an open InsP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.

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Atp Regulation of Recombinant Type 3 Inositol 1,4,5-Trisphosphate Receptor Gating

The Journal of General Physiology ◽

10.1085/jgp.117.5.447 ◽

2001 ◽

Vol 117 (5) ◽

pp. 447-456 ◽

Cited By ~ 31

Author(s):

Don-On Daniel Mak ◽

Sean McBride ◽

J. Kevin Foskett

Keyword(s):

Free Acid ◽

Second Messengers ◽

Hill Coefficient ◽

Open Probability ◽

Isoform Diversity ◽

Recombinant Type ◽

Inositol 1,4,5 Trisphosphate ◽

Patch Clamp Electrophysiology ◽

Type 3

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP]i), here we studied the effects of [ATP]i on the dependence of r-InsP3R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i). As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 μM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (<1 μM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.

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Regulation by Ca2+ and Inositol 1,4,5-Trisphosphate (Insp3) of Single Recombinant Type 3 Insp3 Receptor Channels

The Journal of General Physiology ◽

10.1085/jgp.117.5.435 ◽

2001 ◽

Vol 117 (5) ◽

pp. 435-446 ◽

Cited By ~ 102

Author(s):

Don-On Daniel Mak ◽

Sean McBride ◽

J. Kevin Foskett

Keyword(s):

Mammalian Cells ◽

Single Channel ◽

Channel Gating ◽

Hill Coefficient ◽

Release Channel ◽

Recombinant Type ◽

Inositol 1,4,5 Trisphosphate ◽

Type 3 ◽

Insp3 Receptor

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is an endoplasmic reticulum–localized Ca2+-release channel that controls complex cytoplasmic Ca2+ signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 InsP3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 μM under saturating (10 μM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP3 concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of ∼4. InsP3 activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3–induced Ca2+ release and low gain Ca2+–induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

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Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice

AJP Renal Physiology ◽

10.1152/ajprenal.00152.2017 ◽

2017 ◽

Vol 313 (6) ◽

pp. F1243-F1253 ◽

Cited By ~ 21

Author(s):

Minolfa C. Prieto ◽

Virginia Reverte ◽

Mykola Mamenko ◽

Marta Kuczeriszka ◽

Luciana C. Veiras ◽

...

Keyword(s):

Renal Function ◽

Collecting Duct ◽

Ang Ii ◽

Open Probability ◽

Sodium Transporters ◽

Prorenin Receptor ◽

Active Channels ◽

The Impact ◽

Renal Responses ◽

Stimulation Of

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg−1·min−1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

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Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron

AJP Renal Physiology ◽

10.1152/ajprenal.00606.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. F1105-F1115 ◽

Cited By ~ 36

Author(s):

Oleg Zaika ◽

Mykola Mamenko ◽

Roger G. O'Neil ◽

Oleh Pochynyuk

Keyword(s):

Critical Role ◽

Na Channel ◽

Open Probability ◽

Kinin System ◽

Renal Kallikrein ◽

Subsequent Activation ◽

Patch Clamp Electrophysiology ◽

B2 Receptors ◽

Renal Tubular ◽

Kallikrein Kinin System

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na+ channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability ( Po) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg9-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC Po when Gq/11 was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of Gq/11-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI( 4 , 5 )P2 levels. However, inhibition of Ca2+ pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca2+]i as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC Po in mammalian ASDN via stimulation of B2 receptors and following depletion of PI( 4 , 5 )P2, but not increases in [Ca2+]i.

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Collecting duct-specific endothelin B receptor knockout increases ENaC activity

AJP Cell Physiology ◽

10.1152/ajpcell.00301.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. C188-C194 ◽

Cited By ~ 41

Author(s):

Vladislav Bugaj ◽

Elena Mironova ◽

Donald E. Kohan ◽

James D. Stockand

Keyword(s):

Blood Pressure ◽

Collecting Duct ◽

Endothelin Receptors ◽

Sodium Retention ◽

Wild Type ◽

Open Probability ◽

Endothelin System ◽

Etb Receptor ◽

Endothelin B ◽

Patch Clamp Electrophysiology

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na+ excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na+ regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na+ intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na+ regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na+ feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na+ retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

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Isolation of a recombinant type 3/type 2 poliovirus with a chimeric capsid VP1 from sewage in Shandong, China

Virus Research ◽

10.1016/j.virusres.2010.02.014 ◽

2010 ◽

Vol 150 (1-2) ◽

pp. 56-60 ◽

Cited By ~ 19

Author(s):

Zexin Tao ◽

Haiyan Wang ◽

Aiqiang Xu ◽

Yong Zhang ◽

Lizhi Song ◽

...

Keyword(s):

Recombinant Type ◽

Type 3

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Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.11.8918899 ◽

1996 ◽

Vol 44 (11) ◽

pp. 1243-1250 ◽

Cited By ~ 19

Author(s):

L M Matovcik ◽

A R Maranto ◽

C J Soroka ◽

F S Gorelick ◽

J Smith ◽

...

Keyword(s):

Protein Kinase ◽

Pancreatic Acinar Cells ◽

Apical Region ◽

Cam Kinase Ii ◽

Cam Kinase ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein ◽

Type 3 ◽

Insp3 Receptor

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

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Single-Channel Properties in Endoplasmic Reticulum Membrane of Recombinant Type 3 Inositol Trisphosphate Receptor

The Journal of General Physiology ◽

10.1085/jgp.115.3.241 ◽

2000 ◽

Vol 115 (3) ◽

pp. 241-256 ◽

Cited By ~ 63

Author(s):

Don-On Daniel Mak ◽

Sean McBride ◽

Viswanathan Raghuram ◽

Yun Yue ◽

Suresh K. Joseph ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Single Channel ◽

Calcium Influx ◽

Endoplasmic Reticulum Membrane ◽

Release Channel ◽

Cellular Expression ◽

Recombinant Type ◽

Specific Regulation ◽

Trisphosphate Receptor ◽

Type 3

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca2+ signaling in most cell types. A family of InsP3Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP3Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP3R-3), a widely expressed isoform also implicated in plasma membrane Ca2+ influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP3R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP3R-3 are strikingly similar to those of Xenopus type 1 InsP3R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP3R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP3R isoforms is a mechanism to modify the temporal and spatial features of [Ca2+]i signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP3Rs that have relatively similar Ca2+ permeation properties.

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Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action

Cells ◽

10.3390/cells10082101 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2101

Author(s):

Samantha C. Salvage ◽

Esther M. Gallant ◽

James A. Fraser ◽

Christopher L.-H. Huang ◽

Angela F. Dulhunty

Keyword(s):

Atrial Fibrillation ◽

Ryanodine Receptor ◽

Polymorphic Ventricular Tachycardia ◽

Sodium Currents ◽

Wild Type ◽

Open Probability ◽

Active Channels ◽

Separate Activation ◽

Low Activity ◽

Cardiac Ryanodine Receptor

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.

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