Nonhydrolyzable Analogues of GTP Activate a New Na+ Current in a Rat Mast Cell Line

Parekh, Anant B.

doi:10.1074/jbc.271.38.23161

Cited by 13 publications

(14 citation statements)

References 21 publications

(16 reference statements)

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“…Dialysis with 250 M GTP␥S did not affect slow inactivation either (inactivation of 77 Ϯ 9%). Later on in these same cells (after a 5-10-min recording), the GTP␥S-dependent Na ϩ current activated (14), demonstrating that the GTP␥S was active under these conditions.…”

Section: Investigation Into the Mechanism Of Camentioning

confidence: 74%

Slow Feedback Inhibition of Calcium Release-activated Calcium Current by Calcium Entry

Parekh

1998

Journal of Biological Chemistry

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102

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In many nonexcitable cells, depletion of the inositol 1,4,5-trisphosphate-sensitive store activates Ca 2؉ influx, a process termed store-operated Ca 2؉ entry. In rat basophilic leukemia cells, emptying of the stores activates a highly selective Ca 2؉ release-activated Ca 2؉ current (CRAC), I CRAC . We have recently found that I CRAC activates in an essentially all-or-none manner when the current is evoked by receptor stimulation, dialysis with inositol 1,4,5-trisphosphate via the patch pipette, or through the Ca 2؉ ATPase inhibitor thapsigargin (Parekh, A. B., Fleig, A., and Penner, R. (1997) Cell 89, 973-980). Regulatory mechanisms must therefore operate to control the overall amount of Ca 2؉ that enters through CRAC channels. Such mechanisms include membrane potential and protein kinase C. In the present study, we have investigated additional inhibitory pathways that serve to determine just how much Ca 2؉ can enter through I CRAC . We have directly measured the current using the whole cell patch clamp technique. We report the presence of a slow Ca 2؉ -dependent inactivation mechanism that curtails Ca 2؉ entry through CRAC channels. This inactivation mechanism is switched on by Ca 2؉ entering through CRAC channels, and therefore constitutes a slow negative feedback process. Although it requires a rise in intracellular Ca 2؉ for activation, it maintains CRAC channels inactive even under conditions that lower intracellular Ca 2؉ levels. The inactivation mechanism does not involve store refilling, protein phosphorylation, G proteins, nor Ca 2؉ -dependent enzymes. It accounts for up to 70% of the total inactivation of I CRAC , and therefore appears to be a dominant inhibitory mechanism. It is likely to be an important factor that shapes the profile of the Ca 2؉ signal in these nonexcitable cells.

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Section: Investigation Into the Mechanism Of Camentioning

confidence: 74%

Slow Feedback Inhibition of Calcium Release-activated Calcium Current by Calcium Entry

Parekh

1998

Journal of Biological Chemistry

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102

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“…First, it increased the electrical driving force for Ca 2+ entry through CRAC channels. Second, the Na + current activated by GTP[γ-S] is largely inactive at this potential [20]. The slowly developing Na + current would interfere with the capacitance measurements by inducing a large increase in membrane conductance.…”

Section: Methodsmentioning

confidence: 99%

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

Artalejo¹,

Jc²,

Ab³

1998

Pflügers Arch

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Ca 2+ -dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 µM free Ca 2+ and 300 µM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca 2+ (225 nM to 10 µM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca 2+ buffering (0.1 mM EGTA), activation of Ca 2+ -release-activated Ca 2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP 3 ) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca 2+ ATPases were inhibited by thapsigargin, InsP 3 and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca 2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca 2+ influx because it was effectively suppressed when external Ca 2+ was removed. Our results demonstrate that I CRAC represents an important source of Ca 2+ for triggering a secretory response.

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“…Similar observations were made on planar lipid bilayers containing cloned epithelial sodium channels (4, 7). In addition, interaction of actin with epithelial channels was reported to modulate considerably the intrinsic channel characteristics including conductance and selectivity.Along with epithelial amiloride-sensitive sodium channels, novel non-voltage-gated sodium-selective channels insensitive to amiloride (up to 100 M) and to tetrodotoxin have been described in different non-excitable cells, particularly in vascular smooth muscle cells (8), macrophages (9), and carcinoma (10) and leukemia cells (11)(12)(13). In these studies, single current measurements in different patch configurations showed that conductance, selectivity, and gating properties of the novel family of sodium channels proved to be very similar to those of the epithelial channels (14).…”

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confidence: 99%

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

Negulyaev¹,

Khaitlina²,

Hinssen³

et al. 2000

Journal of Biological Chemistry

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The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in nonexcitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 M [Ca 2؉ ] i . The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mM MgCl 2 to induce actin polymerization. In the absence of MgCl 2 , addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.Functional coupling between channel proteins and the cortical cytoskeleton may play a key role in membrane ion transport and cellular signaling. Involvement of F-actin in ion channel functioning has been established and studied extensively in polarized epithelial cells (1-7). Specifically, several lines of evidence revealed an association between the amiloride-sensitive sodium channels and the actin-based cytoskeleton in renal epithelia. Indirect immunofluorescence and confocal microscopy demonstrated that sodium channels in the apical membrane colocalize with actin, spectrin (fodrin), and ankyrin (5, 6). Electrophysiological studies on epithelial A6 cells showed that disruption of actin microfilament networks by cytochalasin D induced sodium channel activity both in cell-attached and excised patches (1). Similar effects were observed in the presence of actin or actin-gelsolin complexes added to the cytoplasmic side of excised inside-out patches, whereas the actin-DNase I complexes did not activate sodium channels. These results were explained by a model suggesting that the channels are activated by short actin filaments produced either by severing of endogenous long filaments with cytochalasin or by assembly from monomeric actin during spontaneous or gelsolin-mediated polymerization (1). Similar observations were made on planar lipid bilayers containing cloned epithelial sodium channels (4, 7). In addition, interaction of actin with epithelial channels was reported to modulate considerably the intrinsic...

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Nonhydrolyzable Analogues of GTP Activate a New Na+ Current in a Rat Mast Cell Line

Cited by 13 publications

References 21 publications

Slow Feedback Inhibition of Calcium Release-activated Calcium Current by Calcium Entry

Slow Feedback Inhibition of Calcium Release-activated Calcium Current by Calcium Entry

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

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