Intracellular Ca2+ plays a central role in controlling lymphocyte function. Nonetheless, critical gaps remain in our understanding of the mechanisms that regulate its concentration. Although Ca2+-release-activated calcium (CRAC) channels are the primary Ca2+ entry pathways in T cells, additional pathways appear to be operative in B cells. Our efforts to delineate these pathways in primary murine B cells reveal that Ca2+-permeant nonselective cation channels (NSCCs) operate in a cooperative fashion with CRAC. Interestingly, these non-CRAC channels are selectively activated by mechanical stress, although the mechanism overlaps with BCR-activated pathways, suggesting that they may operate in concert to produce functionally diverse Ca2+ signals. NSCCs also regulate the membrane potential, which activates integrin-dependent binding of B cells to extracellular matrix elements involved in their trafficking and localization within secondary lymphoid organs. Thus, CRAC and distinct Ca2+ permeant NSCCs are differentially activated by the BCR and mechanical stimuli and regulate distinct aspects of B cell physiology.

Engagement of the BCR triggers changes in intracellular Ca2+ concentration that initiate specific and tightly regulated adaptive immune functions. Furthermore, distinct patterns of Ca2+ signaling in B cells produced by Ag dictate alternative programs of transcription factor activation and thereby distinct cell fates. Innate stimuli, such as osmotic and oxidative stress, also elevate calcium (1). The specific mechanisms by which these and other BCR-independent stimuli initiate Ca2+ signals, the pathways (channels and stores) that control BCR-independent Ca2+ signaling in primary B cells, and the consequences of activating these pathways represent a significant gap in our understanding of how B cell activation is regulated.

Studies to date suggest that dynamic changes in free cytosolic Ca2+ triggered by Ag receptor engagement principally reflect the interplay between inositol 1,4,5-triphosphate (IP3)2-regulated intracellular Ca2+ stores and Ca2+ entry through “store-operated” Ca2+-release-activated calcium (CRAC) channels (reviewed in Ref. 2). CRAC channels are required for Ag-mediated responses of T cells; however, a similar absolute requirement for CRAC channels in B cells is an open question, given studies of patients with a defect in CRAC activity whose B cells (but not T cells) appear to be capable of mounting normal immune responses (3, 4, 5). Alternative Ca2+ entry pathways in B cells could allow for physical segregation of BCR-dependent and -independent Ca2+ signaling.

In this study, we detail our efforts to further elucidate the pathways that regulate calcium entry into primary mammalian B lymphocytes by addressing the mechanisms and consequences of both Ag receptor-mediated and mechanically induced calcium signaling. Specifically, we asked whether Ca2+ signaling in primary B cells might be regulated by Ca2+ permeant channels other than CRAC. Among the potential candidates for such a role are nonselective cation channels (NSCCs), including a growing number of the transient receptor potential (TRP) family members. More than 22 mammalian TRP genes have been identified in six related protein families, including the TRPC (canonical), TRPV (vanilloid receptor-like), and TRPM (melastatin-like) types (reviewed in Refs. 6 and 7). These NSCCs include members that can be activated by osmotic, temperature, and oxidative stresses. Several, including TRPM2 (8), TRPC1, TRPC3, TRPC4, and TRPC6, have been identified in lymphocytes (9). TRPC1 and TRPC3 have been implicated in Ag receptor-induced (phospholipase C (PLC)-dependent) Ca2+ release from intracellular stores and Ca2+ entry in avian DT40 B cells and Jurkat cells, respectively (10, 11). In addition, (PLC-mediated) phosphatidylinositol hydrolysis (12) regulates TRPM7 channels, which are also found in T lymphocytes. Thus, although PLC activity is central to the activation of CRAC and a number of TRP channels, the physiological activation mechanism and immunological role of NSCCs in primary B lymphocytes represents a gap in our understanding.

Data presented in this manuscript demonstrate that shear and osmotic stress elevate cytosolic Ca2+ in splenic B cells in part due to activation of Ca2+ permeant NSCCs. NSCCs activated by osmotic stress are not store-operated, although mechanical stress does deplete Ca2+ stores and activate CRAC-like currents. The signal transduction pathway responsible for activating NSCCs involves PLCγ-2 and its product diacylglycerol (DAG). This is consistent with previous studies of lymphocytes that suggest DAG is capable of inducing store-independent Ca2+ influx (13). Paradoxically, BCR engagement, which also activates PLCγ-2 and elicits CRAC-like currents, does not activate NSCCs, but instead suppresses NSCC activation and functions of B cells uniquely regulated by NSCCs. Together, our results point to a novel paradigm of Ca2+ signaling that reflects the cooperative regulation of CRAC and NSCC activity by BCR and mechanical sensory pathways that dictate B cell responses in vivo.

Spleens from C57BL/6 mice were placed into complete RPMI 1640 medium, mechanically disrupted, and filtered through sterile nylon mesh (70-μM pore diameter) to remove large debris. Filtered cell suspensions were incubated with a mixture of mAbs against CD4, CD8, CD11b, GR-1, and Ter119 expressed on erythrocytes. Ab-stained cells, including erythrocytes, were removed by immunomagnetic depletion (Stemcell Technologies). Viable cells were recovered and washed twice in RPMI 1640 medium before use in experiments. The purity of nonstained B lymphocytes was determined by flow cytometry. These methods yielded a final population that contained at least 95% B220+ cells.

Patch clamp measurements were performed on purified murine B cells (see above), or cells were identified visually by negative immunofluorescence (CD3 negative) in the microscope recording chamber. Patch pipettes were fabricated with a 4- to 6-MΩ tip resistance (Sutter Instruments) from borosilicate glass and were back-filled with appropriate internal solution. Liquid junction potentials were calculated and were corrected manually with the patch clamp amplifier or post analysis. After formation of gigaohm seals (5–10 GΩ), cells were lifted off the chamber bottom and held at −70 mV, unless indicated otherwise. Command potentials were generated using an EPC-9 or EPC-10 patch clamp amplifier and currents were acquired, stored, and analyzed using PulseFit software. Single channel amplitude frequency analysis was performed using TAC software (Bruxton). Data sampled at 3 kHz were recorded on computer disk and further filtered at 500 Hz during analysis. The instantaneous current reversal potential was typically determined by applying 160-mV voltage ramps (−80 mV to + 80 mV, 200 milliseconds) to the patched cell before and after stimulation. All ramp currents shown were leak corrected by subtracting ramp currents obtained immediately after establishment of a stable whole cell recording. Although ramps were typically applied every 5–10 s, they are filtered from macroscopic current recordings for ease of visualization and are plotted separately.

Membrane permeant drugs were typically applied by direct addition to the bath; however, membrane impermeant inhibitors and mAbs used to test the role of specific enzymes or isoforms of enzymes in stimulus-channel coupling were dialyzed into single cells from the patch clamp recording microelectrode during whole cell recordings. BCR engagement was initiated with soluble goat anti-mouse IgM F(ab′)2 μ-chain specific Ab (Jackson ImmunoResearch Laboratories). The PLC inhibitor U73122, the inactive control compound U73433, and the protein kinase C (PKC) inhibitor peptides 19-39 and RO-31822 were obtained from EMD Biosciences. Polyclonal rabbit anti-PLCγ-1 and -PLCγ-2 Abs were obtained from Upstate Biotechnologies, and rabbit Ig was obtained from Jackson ImmunoResearch Laboratories. Thapsigargin (Tg) and other chemicals used to formulate recording solutions were obtained from Sigma-Aldrich. The standard extracellular bath solution used to measure both TRP and CRAC currents contained 155 mM Na+ gluconate, 2 mM Ca2+ gluconate, 1 mM MgSO4, 10 mM Na+ HEPES, and 10 mM glucose adjusted to pH 7.4 with NaOH. A low Na+ Ringers solution used to determine the cation permeability of channels consisted of 155 mM N-methyl-d-glucamine (NMDG) gluconate, 4.5 mM K+ gluconate, 2 mM Ca2+ gluconate, 1 mM MgSO4, 10 mM Na+ HEPES, and 10 mM d-glucose (pH 7.4). In some experiments, the low Na+ solutions contained 125 mM NMDG-gluconate and 30 mM NaCl. Hypotonic (200 mOsm) solutions, except those used to define the relative Na+:Ca2+ permeability (see below), were all formulated by omitting 50 mM cation (Na+ or other impermeant cation) and its anion pair from external solutions. The standard pipette solution used to measure TRP and CRAC currents contained 155 mM Cs-methanesulfonate, 6 mM MgSO4, 1 mM EGTA, 0.25 mM Ca2+ gluconate (62 nM free Ca2+), and 10 mM Cs HEPES (pH adjusted to 7.4). To determine the relative Ca2+ vs Na+ permeability of channels, the bath solution contained 100 mM NMDG chloride, 30 mM Ca2+ chloride, 10 mM glucose, 20 mM HEPES adjusted to pH 7.4, and the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 100 μM). The pipette solution contained 137 mM NMDG chloride, 10 mM Na+ chloride, 20 mM HEPES, adjusted to pH 7.2. NPPB (100 μM) was added to block chloride channels. Hypotonic external solutions used to activate TRP currents during measurements of the relative Ca2+:Na+ permeability contained 50 mM NMDG-chloride, 30 mM Ca2+ chloride, and 20 mM HEPES, 10 mM glucose as well as NPPB. The relative permeability (PCa2+/PNa+) was calculated using the measured reversal potential in the equation Erev = RT/2F ln(4PCa2+[Ca2+]o/PNa+[Na+]i), as previously described (14). The osmolarity of all solutions was confirmed using a freezing point depression osmometer.

The on-cell configuration of the patch clamp was used to obtain single channel recordings from cells bathed in 155 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM K+-HEPES, and 10 mM glucose adjusted to pH 7.2, using a pipette solution that contained 155 mM K+ gluconate, 10 mM K+ HEPES, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 100 μM NPPB (to block Cl channels), and 100 nM charybdotoxin (to block voltage-gated and Ca2+ activated channels).

Lymphocytes were loaded with the cell permeant calcium indicator fura 2-AM (3.0 μM; Molecular Probes, Eugene, OR) in RPMI 1640 medium for 15 min at room temperature (25°C). Cell suspensions were placed into the recording chamber on an inverted fluorescence microscope (Nikon) and allowed to adhere to Poly-l-lysine (100 μg/ml; Sigma-Aldrich)-treated coverslips for 5 min in a solution that contained 155 mM NaCl, 4.5 KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). Excess fura 2-AM was removed by perfusing the chamber with extracellular solution. Intracellular Ca2+ was measured by digital imaging microscopy as previously described (15)

B lymphocytes were identified as described above, and membrane voltage (Vm) was measured directly using the current clamp configuration of the nystatin-perforated patch clamp technique as previously described (15). Cells were bathed in 155 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 4.5 mM KCl, 10 mM Na+ HEPES, and 10 mM glucose adjusted to pH 7.4 with NaOH. A low Na+ ringers solution used to define the contribution of NSCCs in membrane potential regulation by mechanical stimulation consisted of 155 mM NMDG-Cl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM Na+ HEPES, and 10 mM d-glucose (pH 7.2). The pipette solution contained 155 mM KCl, 2 mM MgSO4, 1 mM EGTA, 0.25 mM CaCl2 (62 nM free Ca2+), 10 mM HEPES, and nystatin (100 μg/ml) (pH adjusted to 7.2). The membrane potential was measured in viable cells using patch clamp in the current-clamp recording mode. The role of NSCCs (defined in voltage-clamp experiments) in regulating the steady-state Vm was determined by activating cells mechanically or with 1-oleoyl-2-acetyl-sn-glycerol (OAG) and then removing extracellular Na+ to examine its role in stimulus-induced changes in Vm.

Total cellular RNA was prepared according to the acid guanidinium method (16). cDNA synthesis with Superscript II reverse transcriptase (Invitrogen Life Technologies) PCR was used to amplify cDNA prepared from B cell RNA. Unique complementary oligonucleotide primer pairs were designed for each mammalian transient receptor potential gene TRPC1–TRPC7, TRPV1–TRPV6, and TRPM1–TRPM8 (the sequence of these primers can be obtained by contacting the corresponding author). The specificity of each of these oligonucleotide primer pairs was confirmed in test PCRs using mouse genomic DNA or cDNA clone as a template (data not shown). PCR products were separated by electrophoresis in a 2% agarose gel and were identified by ethidium staining. The identity of each PCR product of expected size was confirmed by sequencing.

Protein immunoprecipitations and Western analysis were performed using standard methods. Immunoprecipitation of whole cell lysates was performed with anti-phosphotyrosine (clone 4G10), and phospho-PLC was detected by probing blots with anti-PLCγ-1 or -PLCγ-2 Abs (Upstate Biotechnologies). HRP-conjugated anti-goat and anti-rabbit secondary Abs were obtained from Jackson ImmunoResearch Laboratories.

Murine integrin avidity changes were assessed using an ICAM-1 and VCAM-1 adhesion assay similar to that previously described (17) with the following modifications. Immunolon 4 plates (Costar) were treated with anti-human Fcγ (10 μg/ml in PBS; Jackson ImmunoResearch Laboratories) overnight at 37°C, and then wells were rinsed with PBS. Plates were blocked with 1% BSA in PBS for 1 h at 37°C to prevent nonspecific binding, after which plates were washed twice with PBS. Murine ICAM-1 (human) Fc fusion protein (10 μg/ml; R&D Systems), murine VCAM-1 (human) Fc fusion protein (20 μg/ml; R&D Systems), or human IgG (10 μg/ml; Jackson ImmunoResearch Laboratories) was added to wells for 1.5 h at 37°C followed by two PBS washes. Splenocytes (4 × 106/ml, 100 μl) in complete RPMI 1640 and an additional 100 μl of complete medium containing indicated stimuli were added to each well. Cultures were incubated for 0.5 h at 37°C. Unbound cells and media were removed by washing wells extensively with PBS. Adherent cells were subsequently removed from wells by incubating with complete RPMI 1640 containing 5 mM EDTA (200 μl) for 15 min at 4°C, enumerated, stained with anti-B220, and analyzed by flow cytometry to determine the absolute number of B cells recovered from each well. Results are expressed as the percentage of total B cells recovered from plates after subtraction of nonspecific binding values obtained from wells treated with human IgG.

In T cells, fluid shear force has been found to activate integrins necessary for T cell adhesion and transit, leading us to speculate that mechanical force may do so in part by elevating intracellular Ca2+. To examine the effect of shear force on lymphocytes, we applied a high-pressure stream of extracellular fluid onto unseparated splenic lymphocytes with an angled microinjection pipette and found that, indeed, fluid shear force mobilizes intracellular Ca2+ in a subpopulation of cells (Fig. 1,A). Upon closer inspection of the effect, we were surprised to find that this shear-induced response was restricted to B lymphocytes within the fluid stream (determined by post-experimental staining of cells with anti-B220 Ab). Ca2+ mobilization reflected intracellular release as well as extracellular influx (data not shown). Because fluid shear triggered Ca2+ entry, we used the patch clamp to define currents produced by its movement into the cell. Fluid shear elicited a relatively large inward ion current (−253 ± 30 pA, n = 17, VH = −80 mV) within 77% of B cells tested (Fig. 2,B), and the current reversal potential (inset; Erev = −1.6 ± 1.0 mV, n = 17 cells) indicated near equal permeability to intracellular and extracellular cations. A reduction in extracellular sodium concentration (Na+ replaced with impermeant 135 mM NMDG) caused a negative shift in the reversal potential (Erev = −48 ± 6 mV, n = 6; Fig. 2 B, right panel). Pressure-induced currents were also blocked >90% by 10 μM lanthanum (data not shown). Thus, fluid shear elicits calcium elevations and NSC currents in B cells.

FIGURE 1.

Fluid shear stress elevates cytosolic Ca2+ and elicits an NSC current in B cells. A, Pseudocolor image of fura-2 fluorescence in lymphocytes. Fluid shear elevated Ca2+ in a subpopulation of cells. The micropipette used to apply a high-pressure fluid stream is seen to the right of the image, and the central point of fluid impact is evident toward the left. Immunofluorescence staining performed in situ after measuring Ca2+ indicated that Ca2+ was elevated (yellow or red) only in B220+, and not CD4+ or CD8+ lymphocytes (phenotyping not shown). Several cells not within the fluid stream that exhibit elevated calcium were activated before application of fluid pressure. B, Patch clamp recording of steady-state current elicited by fluid pressure in B cell at −60 mV holding potential. The Erev (inset) of these currents measured at peak amplitude in normal (155 mM) (a) external Na+ was ∼0 mV and shifted to ∼−60 mV in low Na+ (30 mM) (b) external solution. This current is representative of measurements obtained from 28 individual cells.

FIGURE 1.

Fluid shear stress elevates cytosolic Ca2+ and elicits an NSC current in B cells. A, Pseudocolor image of fura-2 fluorescence in lymphocytes. Fluid shear elevated Ca2+ in a subpopulation of cells. The micropipette used to apply a high-pressure fluid stream is seen to the right of the image, and the central point of fluid impact is evident toward the left. Immunofluorescence staining performed in situ after measuring Ca2+ indicated that Ca2+ was elevated (yellow or red) only in B220+, and not CD4+ or CD8+ lymphocytes (phenotyping not shown). Several cells not within the fluid stream that exhibit elevated calcium were activated before application of fluid pressure. B, Patch clamp recording of steady-state current elicited by fluid pressure in B cell at −60 mV holding potential. The Erev (inset) of these currents measured at peak amplitude in normal (155 mM) (a) external Na+ was ∼0 mV and shifted to ∼−60 mV in low Na+ (30 mM) (b) external solution. This current is representative of measurements obtained from 28 individual cells.

Close modal
FIGURE 2.

Osmotic stress elevates cytosolic Ca2+ and elicits an NSC current in B cells. A, Hypotonic stress (200 mOsm) triggered an increase in cytoplasmic Ca2+ in primary B cells. B, Patch clamp recording of inward whole cell current elicited by hypotonicity in a B cell held at −70 mV. Current voltage analysis (a, inset) of hypotonicity induced current reversed direction at ∼0 mV. Switch to Na+-free hypotonic external solution (105 mM to 0 mM) produces a negative shift in Erev. In this example, in the absence of extracellular Na+, the Erev shifted to ∼−70 mV (b) and complete attenuation of the steady-state current at VH. The hypotonicity-activated current was blocked by La3+ (100 μM). This current recording shown is representative of similar measurements obtained from >84 individual cells.

FIGURE 2.

Osmotic stress elevates cytosolic Ca2+ and elicits an NSC current in B cells. A, Hypotonic stress (200 mOsm) triggered an increase in cytoplasmic Ca2+ in primary B cells. B, Patch clamp recording of inward whole cell current elicited by hypotonicity in a B cell held at −70 mV. Current voltage analysis (a, inset) of hypotonicity induced current reversed direction at ∼0 mV. Switch to Na+-free hypotonic external solution (105 mM to 0 mM) produces a negative shift in Erev. In this example, in the absence of extracellular Na+, the Erev shifted to ∼−70 mV (b) and complete attenuation of the steady-state current at VH. The hypotonicity-activated current was blocked by La3+ (100 μM). This current recording shown is representative of similar measurements obtained from >84 individual cells.

Close modal

In subsequent experiments aimed at further understanding the physiological relevance of this mechanically activated Ca2+ signaling pathway, we discovered that other forms of stress, including osmotic stress, produced a similar response in B cells. Lymphocytes subjected to hypotonic stress swell due to water entry, and this causes the membrane to stretch. Subsequent activation of Cl and K+ channels facilitates solute efflux, and cells return to a prestress volume within ∼10 min (18, 19). Although osmotic stress has been shown to elevate Ca2+ in B cell lines (4), its consequences on primary B cells have not been investigated. To examine this, it was necessary to avoid the common procedure involving hypotonic lysis to remove RBCs from splenocyte suspensions. Consequently, an Ab-mediated immunomagnetic method was used to deplete RBCs from these preparations (see Materials and Methods).

Like fluid shear, hypotonic stress (200 milliosmolar (mOsm); normal murine serum osmolarity = 300 mOsm) elicited a Ca2+ increase, which rapidly reached a peak concentration (145 nM, n > 200 cells, three similar experiments) and then decayed to an elevated (mean = 85 nM) steady-state level, from which periodic spikes in Ca2+ continued to develop (Fig. 2 A). The initial rise in Ca2+ occurred after a mean time delay of 53 ± 13 s (range = 30–65 s). In Ca2+-free external solution, only an initial transient elevation in cytoplasmic Ca2+ was observed, indicating that osmotic stress induces Ca2+ release from intracellular stores. A secondary Ca2+ increase, indicative of influx, was produced when cells were subsequently superfused with Ca2+ containing external solution (data not shown).

Because hypotonicity triggered extracellular Ca2+ influx, we used the patch clamp to define the currents associated with Ca2+ entry. Hypotonic challenge activated an inward current (−56 ± 4 pA, n = 84 cells; Fig. 2,B) at negative holding potentials (VH = −70 mV) under conditions that precluded K+ and Cl currents (see Materials and Methods), also activated by hypotonicity in lymphocytes (20). The time between hypotonic challenge and current activation varied significantly, ranging from several seconds to more than a minute in some instances. This variable delay was a surprising, but consistent finding for both shear-activated and osmotically activated currents. Although hypotonicity-induced NSC currents sometimes exhibited fluctuations in amplitude, they typically reached a sustained steady-state level within 60 s after the inward current first appeared. In this example, upon reintroduction of extracellular Na+, the current was larger than that achieved before ion substitution and larger in amplitude than the average, suggesting that Na+-dependent attenuation could limit the NSCC amplitude or activation. In some experiments, currents subsequently decayed to prestimulation levels in the presence of hypotonic stress, although more typically channels would close only upon removal of this stress. Hypotonicity-induced currents in B cells reversed direction at −2 ± 1.5 mV (n = 84 cells), which was similar to those elicited with shear force, and the reversal potential shifted to a more negative value (−45 ± 5 mV, n = 6) in low Na+ (30 mM Na+, NMDG substituted) external solution as expected for a cation channel. In the example shown (Fig. 2,B, inset), the negative shift in Erev was greater than this average value because all Na+ was removed from the bath (0 mM Na+). Hypotonicity-induced NSC currents were sensitive to La3+ (IC50 = 6 μM; see Fig. 4E) and 2-aminoethyldiphenyl borate (2-APB) (IC50 = ∼1.5 μM), each of which also blocks CRAC channels (21), but were insensitive to the CRAC channel inhibitor SKF-96365 (50 μM; data not shown).

FIGURE 4.

NSCCs in B cells are not store operated. A, Simultaneous measurement of intracellular Ca2+ and whole cell currents in single B cells. Tg (1 μM) elevated [Ca2+]i (bottom trace) and activated a small-amplitude inward current (top trace) in Ca2+-free, Mg2+-containing external solution. B, I-V analysis of Tg-induced current (Ab) demonstrates strong inward rectification and a positive reversal potential (∼50 mV). In DVF bath solution (A, filled bar), the current amplitude increased, but the current reversal potential did not change (Ba, ∼50 mV). This Tg-induced current inactivated within ∼4 min and no additional current developed in the presence of Tg; however, hypotonic stress (200 mOsm) subsequently elicited an inward current (VH = −70 mV). This hypotonicity-induced current exhibited a linear I-V relationship and Erev = ∼0 mV (c). These dual current and Ca2+ recordings are typical of six similar experiments. C, OAG (200 μM) induces oscillatory changes in [Ca2+]i in the presence (left), but not the absence (right), of extracellular Ca2+. D, OAG-induced NSC currents exhibit a linear I-V relationship (b-a, inset), and removal of extracellular Na+ decreased the current amplitude at VH = −60 mV and caused a negative shift in the current-reversal potential Erev (c-a, inset). The example of an OAG current shown exhibits a mild degree of fluctuation, although in some cells OAG-induced currents exhibited more prominent low-amplitude fluctuations in amplitude or no fluctuations at all. Ramp currents were leak subtracted as described in Materials and Methods. E, Hypotonicity-induced (□) and OAG-induced (○) currents exhibit identical sensitivity to La3+ inhibition. The peak current in the presence of indicated La3+ concentrations is plotted against the peak steady-state current in the absence of blocker for each individual cell, and each point is the mean (± SD) of at least three separate measurements. F, Tg-sensitive Ca2+ stores are not required for activation of NSC currents by OAG. Intracellular release of Ca2+ from stores with Tg (1 μM) activated a CRAC-like current (a; small inward rectifying current, positive Erev, see I-V to right). DVF solution increased the current amplitude 4- to 5-fold (b, right), but induced no change in reversal potential or rectification properties (see inset at right). After 5 min of Tg treatment, OAG elicited an inward current, which did not rectify and reversed direction at ∼10 mV in this example (right panel). G, On-cell patch clamp recordings of single channel activity elicited by hypotonicity and OAG in membrane patches held at −100 mV. The conductance of hypotonicity and OAG-induced currents (∼30 pS) was determined by amplitude analysis (inset shows distribution for OAG). NSC currents were isolated by treating cells with margatoxin (100 nM) and charybdotoxin (100 nM) to block voltage-gated and calcium-activated K+ currents and with NPPB (100 μM) to block Cl currents.

FIGURE 4.

NSCCs in B cells are not store operated. A, Simultaneous measurement of intracellular Ca2+ and whole cell currents in single B cells. Tg (1 μM) elevated [Ca2+]i (bottom trace) and activated a small-amplitude inward current (top trace) in Ca2+-free, Mg2+-containing external solution. B, I-V analysis of Tg-induced current (Ab) demonstrates strong inward rectification and a positive reversal potential (∼50 mV). In DVF bath solution (A, filled bar), the current amplitude increased, but the current reversal potential did not change (Ba, ∼50 mV). This Tg-induced current inactivated within ∼4 min and no additional current developed in the presence of Tg; however, hypotonic stress (200 mOsm) subsequently elicited an inward current (VH = −70 mV). This hypotonicity-induced current exhibited a linear I-V relationship and Erev = ∼0 mV (c). These dual current and Ca2+ recordings are typical of six similar experiments. C, OAG (200 μM) induces oscillatory changes in [Ca2+]i in the presence (left), but not the absence (right), of extracellular Ca2+. D, OAG-induced NSC currents exhibit a linear I-V relationship (b-a, inset), and removal of extracellular Na+ decreased the current amplitude at VH = −60 mV and caused a negative shift in the current-reversal potential Erev (c-a, inset). The example of an OAG current shown exhibits a mild degree of fluctuation, although in some cells OAG-induced currents exhibited more prominent low-amplitude fluctuations in amplitude or no fluctuations at all. Ramp currents were leak subtracted as described in Materials and Methods. E, Hypotonicity-induced (□) and OAG-induced (○) currents exhibit identical sensitivity to La3+ inhibition. The peak current in the presence of indicated La3+ concentrations is plotted against the peak steady-state current in the absence of blocker for each individual cell, and each point is the mean (± SD) of at least three separate measurements. F, Tg-sensitive Ca2+ stores are not required for activation of NSC currents by OAG. Intracellular release of Ca2+ from stores with Tg (1 μM) activated a CRAC-like current (a; small inward rectifying current, positive Erev, see I-V to right). DVF solution increased the current amplitude 4- to 5-fold (b, right), but induced no change in reversal potential or rectification properties (see inset at right). After 5 min of Tg treatment, OAG elicited an inward current, which did not rectify and reversed direction at ∼10 mV in this example (right panel). G, On-cell patch clamp recordings of single channel activity elicited by hypotonicity and OAG in membrane patches held at −100 mV. The conductance of hypotonicity and OAG-induced currents (∼30 pS) was determined by amplitude analysis (inset shows distribution for OAG). NSC currents were isolated by treating cells with margatoxin (100 nM) and charybdotoxin (100 nM) to block voltage-gated and calcium-activated K+ currents and with NPPB (100 μM) to block Cl currents.

Close modal

NSCCs can exhibit wide variations in cation permeability. Therefore, we next determined the relative Na+ vs Ca2+ permeability of mechanically activated variants in B cells. This assessment has important implications for the physiological functions of these channels and also could help distinguish between multiple NSCCs identified in B cells. This relative permeability was determined under bi-ionic conditions in which Na+ (10 mM) was the only internal membrane permeant cation and Ca2+ (30 mM) was the sole external permeant cation (see Materials and Methods). Although chloride was present in the pipette and bath solutions, B cells were first treated with NPPB (100 μM) to block chloride currents. Under these unusual bi-ionic conditions, hypotonicity activated a current that reversed direction at +14.3 ± 7.7 mV (n = 3; data not shown). The relative Na+ vs Ca2+ permeability (5.0 ± 2.1) calculated using these values indicates that osmotically activated NSCCs are significantly Ca2+ permeant, although they exhibit a greater permeability to Na+.

The biophysical properties of mechanically activated currents are distinct from CRAC currents, although qualitative aspects of mechanically operated Ca2+ signals resemble patterns of Ag receptor-mediated Ca2+ mobilization. For example, both mechanical and BCR stimulation cause Ca2+ release from intracellular stores. Therefore, we asked whether PLC, which links the Ag receptor to intracellular Ca2+ mobilization through the production of IP3, is also activated by mechanical stimulation of B cells.

To test the role of PLC, we first examined the effect of the broad PLC inhibitor U73122 on hypotonicity-induced Ca2+ elevations. We found that U73122, but not the inactive analog U73443, completely attenuated hypotonicity-induced Ca2+ signals (Fig. 3A). U73122 also inhibited activation of NSC currents by hypotonicity and shear force (data not shown). To identify the specific PLC isoform responsible for mechanical signaling, we used a single cell neutralization strategy, which involved dialyzing anti-PLCγ-1 or anti-PLCγ-2 Abs (2 μg/ml) into the cytoplasm from the patch clamp recording pipette (see Materials and Methods) and then examining the amplitude of currents subsequently activated by hypotonic stress in these same cells. This approach is similar to that used by others to identify signal transduction intermediates linking other receptors to the activation of NSCCs in smooth muscle (22) and chloride channels in lymphocytes (23). Our choice of these specific polyclonal Abs was based upon their ability to recognize a three-dimensional conformation of respective PLC isoforms, as indicated by their ability to immunoprecipitate each PLC expressed in murine lymphocytes (see Fig. 3,C and data not shown). Moreover, our findings using this approach are consistent with previous studies, which indicate that PLCγ-2 is the predominant functional isoform in B cells (24). Intracellular dialysis with rabbit serum Ig had no inhibitory effect on osmotic induction of NSC currents (peak current amplitude = 26 ± 9 pA/pF, n = 6), and this was not significantly different from currents in cells treated with anti-PLCγ-1 (Fig. 3,B; 18 ± 5 pA/pF, n = 4). In contrast, intracellular PLCγ-2 neutralization significantly decreased the amplitude of osmotically activated currents (6 ± 2 pA/pF, n = 7; Fig. 3,B, inset). Consistent with this role for PLCγ-2, we also found that hypotonicity increases phospo-PLCγ-2 (Fig. 3 C), but not phospho-PLCγ-1 levels (data not shown) in B cells. Together, these data demonstrate that PLCγ-2 is tyrosine phosphorylated after hypotonic stress and that Abs that specifically recognize PLC-γ2 prevent NSCC activation by hypotonic stress.

FIGURE 3.

PLC activity is required for activation of nonselective cation channels. A, Transient elevation in cytoplasmic Ca2+ elicited by hypotonicity in purified B cells in Ca2+-free bath solution (left panel). The PLC inhibitor U73122 (1 μM) (middle panel) blocked hypotonicity-induced intracellular Ca2+ release, but the structurally similar but inactive analog U73433 (1 μM) did not (right panel). Induction of NSC currents by hypotonicity requires PLCγ-2 activity. B, Polyclonal anti-PLCγ-1, anti-PLCγ-2 Ab, or rabbit Ig (2 μg/ml) used as a control were dialyzed into single cells from the recording pipette for 5 min, and then cells were subjected to hypotonic stress (arrows). An inward current (left) developed in the absence of PLC Abs. Anti-PLCγ-1 treatment had no significant affect on the amplitude of NSC current levels induced with hypotonicity (right, top); however, anti-PLCγ-2 significantly attenuated NSC currents (right, bottom). The mean current amplitude normalized to cell capacitance (size) for each condition is plotted (far right). C, Hypotonicity increased phospho-PLCγ-2 levels. Phosphoproteins were immunoprecipitated with anti-phosphotyrosine (from clone 4G10) and blotted for total PLCγ-2. Phosho-PLCγ-2 levels increased ∼3-fold within the first minute after hypotonic challenge and then decayed to prestimulation levels. Total PLCγ-2 measured in duplicate samples (bottom) demonstrates even sample loading on the gel. A typical example shown is representative of four separate experiments.

FIGURE 3.

PLC activity is required for activation of nonselective cation channels. A, Transient elevation in cytoplasmic Ca2+ elicited by hypotonicity in purified B cells in Ca2+-free bath solution (left panel). The PLC inhibitor U73122 (1 μM) (middle panel) blocked hypotonicity-induced intracellular Ca2+ release, but the structurally similar but inactive analog U73433 (1 μM) did not (right panel). Induction of NSC currents by hypotonicity requires PLCγ-2 activity. B, Polyclonal anti-PLCγ-1, anti-PLCγ-2 Ab, or rabbit Ig (2 μg/ml) used as a control were dialyzed into single cells from the recording pipette for 5 min, and then cells were subjected to hypotonic stress (arrows). An inward current (left) developed in the absence of PLC Abs. Anti-PLCγ-1 treatment had no significant affect on the amplitude of NSC current levels induced with hypotonicity (right, top); however, anti-PLCγ-2 significantly attenuated NSC currents (right, bottom). The mean current amplitude normalized to cell capacitance (size) for each condition is plotted (far right). C, Hypotonicity increased phospho-PLCγ-2 levels. Phosphoproteins were immunoprecipitated with anti-phosphotyrosine (from clone 4G10) and blotted for total PLCγ-2. Phosho-PLCγ-2 levels increased ∼3-fold within the first minute after hypotonic challenge and then decayed to prestimulation levels. Total PLCγ-2 measured in duplicate samples (bottom) demonstrates even sample loading on the gel. A typical example shown is representative of four separate experiments.

Close modal

Because mechanical stimulation activates PLCγ-2 and induces Ca2+ release from stores, we next asked whether mechanically activated NSCCs are store operated. Among the known NSCCs are store-operated as well as store-independent variants. To address the relationship between Ca2+ release from stores and channel activation, we measured membrane currents and intracellular Ca2+ simultaneously in B cells. Ca2+ was mobilized by inhibiting the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) with Tg, causing depletion of Ca2+ from stores (Fig. 4,A, bottom panel). The relatively slow Tg-mediated increase in cytosolic Ca2+ (compared with the rapid increase produced by BCR engagement; see Fig. 5,B) reflects the fact that Tg blocks reuptake of Ca2+ into the endoplasmic reticulum rather than actively inducing its release. Nonetheless, Tg elicited a small-amplitude CRAC-like current (−13 ± 3 pA, VH = −80 mV; Fig. 4,A, top trace) in Ca2+-free ([Mg2+] = 1 mM) solution. This current exhibited a positive reversal potential (57 ± 4 mV, n = 5) and strong inward rectification (Fig. 4,A, right panel), and under fully divalent free conditions (([DVF], [Ca2+]o = 0 mM; [Mg2+]o = 0 mM; intracellular [Mg2+] = 8 mM), the current amplitude increased. Subsequent to Tg-mediated store depletion (∼5 min), hypotonic challenge elicited a biophysically distinct current, which was typically larger in amplitude (mean = −53 ± 14 pA, n = 4 cells) and which reversed direction near 0 mV (Erev = 0.6 ± 3 mV, n = 4; Fig. 4 B). In fact, store depletion (with Tg or intracellular Ca2+ chelators EGTA or BAPTA; data not shown) never elicited an NSC current, suggesting that store depletion is not involved in activating NSCCs in B cells.

FIGURE 5.

Hypotonicity and the BCR activate CRAC-like currents. PKC inhibition blocks hypotonic activation of NSC currents by hypotonicity and reveals an underlying CRAC-like current. A, NSC current activation was blocked by intracellular dialysis with the PKC inhibitor peptide 19-31 (2 μM). Five minutes after application of the inhibitor, hypotonic challenge elicited a current. A typical I-V is shown and exhibits strong inward rectification and a positive reversal potential. Similar results were observed with the membrane permeant PKC inhibitor RO-31822. B, BCR engagement with anti-IgM (50 μg/ml) induced a biphasic change in [Ca2+]i in purified splenic B cells, although NSC currents were never produced by BCR engagement (C), which elicited currents, similar I-V properties, and CRAC, including a positive Erev and small amplitude in divalent bath solution and strong inward rectification similar to CRAC-like currents elicited with Tg (Fig. 3 E). I-V plots in Ca2+ (2 mM) containing (C, top) or DVF external solution (C, bottom) also demonstrate augmentation typical of CRAC under DVF conditions. All ramp currents shown were leak subtracted as described in Materials and Methods. D, Osmotically activated currents are rapidly suppressed by BCR engagement (50 μg/ml anti-IgM). A typical example is shown and is representative of 10 separate measurements.

FIGURE 5.

Hypotonicity and the BCR activate CRAC-like currents. PKC inhibition blocks hypotonic activation of NSC currents by hypotonicity and reveals an underlying CRAC-like current. A, NSC current activation was blocked by intracellular dialysis with the PKC inhibitor peptide 19-31 (2 μM). Five minutes after application of the inhibitor, hypotonic challenge elicited a current. A typical I-V is shown and exhibits strong inward rectification and a positive reversal potential. Similar results were observed with the membrane permeant PKC inhibitor RO-31822. B, BCR engagement with anti-IgM (50 μg/ml) induced a biphasic change in [Ca2+]i in purified splenic B cells, although NSC currents were never produced by BCR engagement (C), which elicited currents, similar I-V properties, and CRAC, including a positive Erev and small amplitude in divalent bath solution and strong inward rectification similar to CRAC-like currents elicited with Tg (Fig. 3 E). I-V plots in Ca2+ (2 mM) containing (C, top) or DVF external solution (C, bottom) also demonstrate augmentation typical of CRAC under DVF conditions. All ramp currents shown were leak subtracted as described in Materials and Methods. D, Osmotically activated currents are rapidly suppressed by BCR engagement (50 μg/ml anti-IgM). A typical example is shown and is representative of 10 separate measurements.

Close modal

Given that hypotonicity induces PLCγ-2-dependent Ca2+ store release, yet Ca2+ release does not activate NSCCs, we wondered whether another product of PLC, namely DAG, might be the trigger for NSCC activation in B cells. To test this hypothesis, we first assessed the ability of DAG to alter intracellular Ca2+ levels. We found that OAG, a potent membrane permeant DAG, produced oscillatory changes in Ca2+ within a significant proportion of B cells (Fig. 4,B; 44 ± 5%, n = 3 experiments with >50 cells/experiment) in the presence but not the absence of extracellular Ca2+ (Fig. 4 C, right panel). Thus, OAG induces Ca2+ influx, but this does not involve Ca2+ release from intracellular stores and suggests a more direct effect on Ca2+ entry channels.

We next determined the properties of membrane currents elicited by OAG in B cells. OAG activated an inward current (−56 ± 4 pA, n = 10 cells; Fig. 4,D) at −60 mV, and the reversal potential shifted from ∼0 mV (−5 ± 3 mV, n = 10) to a more negative value (−43 ± 5 mV, n = 3; Fig. 4,D, inset) in low Na+ (NMDG substituted) external solution, as expected for a sodium permeant channel. OAG-induced NSC currents were sensitive to La3+ (IC50 = 6 μM; see Fig. 4 E) and 2-APB (IC50 = ∼2 μM; data not shown) and were insensitive to SKF-96365 (data not shown).

If the same channel were to underlie currents elicited by both mechanical stimuli and OAG, then OAG activation of NSC currents should be similarly independent of the Ca2+ content of stores. To test this, Ca2+ stores were depleted with Tg, and then cells were stimulated with OAG. Tg alone elicited a small-amplitude (−11 ± 1 pA, n = 4), inwardly rectifying, CRAC-like current (Fig. 4,F), which reversed direction at positive potentials (46 ± 5 mV; Fig. 4,F, right panel). Fully divalent cation-free external solution increased the amplitude of Tg-induced currents ∼5-fold (−61 ± 10 pA, n = 5) with no significant impact on the reversal potential (Erev = 57 ± 7 mV, n = 5). These Tg-induced Na+ currents in divalentcation-free (DVF) solution are similar to those observed above (Fig. 4,B) and are consistent with previous findings that CRAC channels become permeable to Na+ upon removal of external divalent cations (21, 25). Subsequent to Ca2+ store depletion and in the presence of Tg, OAG elicited a distinct inward (−151 ± 50 pA, n = 4; Fig. 4 F) NSC current in Ca2+-containing solution (Erev = −6 ± 4 mV, n = 4), which was similar to NSC currents activated by OAG in cells not treated with Tg. This reversal potential of currents produced by OAG in the presence of Tg was not significantly different from currents elicited by hypotonic stress in the presence of Tg (see above)

OAG-induced (and hypotonicity-induced) NSC currents are distinct from CRAC currents in terms of the peak amplitude, reversal potential, rectification properties, and Ca2+ store-independent activation mechanism. However, OAG- and hypotonicity-induced currents were indistinguishable from one another with respect to their reversal potential, lack of rectification, nonselective cation permeability (Figs. 4,B; 4,D, inset; and 4,F, right panel), and La3+ sensitivity (Fig. 4,E). In addition, under bi-ionic conditions used to determine the Na+:Ca2+ permeability of OAG-induced currents, the reversal potential (Erev = +14.9 ± 6.3 mV, n = 4; data not shown) we measured demonstrated a near identical permeability for Na+ vs Ca2+ (5.7 ± 1.1, n = 4) as hypotonicity-evoked channels. Also, the single channel conductance (Fig. 4 G) of OAG-activated (30.6 ± 1.1 pS, n = 4 cells) and hypotonicity-activated channels (30.8 ± 0.6 pS, n = 9 cells) was found to be similar.

The time delay between OAG application and current activation (124 ± 114 s, n = 44) was highly variable but not statistically different from the delay observed between application of hypotonic solution and current activation (see above). In part this could reflect variability in the time it takes for OAG to reach the cell and/or to accumulate within it after its addition to the bath, or it may reflect variability in the time required for metabolism of OAG to a more proximal activator of NSCCs. Similar delays in NSCC activation by DAG analogues and hypotonicity have been reported for certain TRP family NSCCs. For example, heterologously expressed TRPV4 NSCCs are activated with a variable delay (26) similar to that observed for NSC channels in B cells (27), and, interestingly, B cells express TRPV4 mRNA (see Fig. 8). Our data also suggest that the delayed activation of NSCCs we observe is in part an inherent property of these channels. We found that 5,6-epoxyeicosa-8Z,11Z,14Z-trienoic acid (5,6-EET), a downstream product of OAG metabolism shown to activate TRPV4 (27), activates NSCCs in B cells with a much shorter delay (71.6 ± 21.2 s, n = 6) than OAG. Likewise, the phorbol ester derivative 4-α-phorbol 12,13-didecanoate, which does not activate PKC but directly activates TRPV4 (28), does so with a delay similar to that of 5,6-EET (84.5 ± 48.5 s, n = 6). We hypothesize that the additional delay in activation by OAG or hypotonicity might reflect the time required for metabolism of OAG and accumulation of a downstream intracellular channel agonist. It is also possible that DAG kinases, which are important regulators of DAG signaling and lymphocyte function (29, 30, 31), may contribute to the delay in activation by regulating the time necessary to achieve stimulatory levels of downstream NSCC agonists. Moreover, the activity of this enzyme may vary between cells or preparations.

FIGURE 8.

Molecular identification of mechanically activated currents. RT-PCR analysis of RNA from primary B cells revealed expression of eight TRP mRNA. DNA primer pairs used for amplification were validated on mouse genomic DNA or with cDNA prepared using tissue from which each clone was originally isolated (data not shown). Negative controls were performed on identical mRNA preparations to exclude false positive results due to genomic DNA contamination (data not shown).

FIGURE 8.

Molecular identification of mechanically activated currents. RT-PCR analysis of RNA from primary B cells revealed expression of eight TRP mRNA. DNA primer pairs used for amplification were validated on mouse genomic DNA or with cDNA prepared using tissue from which each clone was originally isolated (data not shown). Negative controls were performed on identical mRNA preparations to exclude false positive results due to genomic DNA contamination (data not shown).

Close modal

The larger mean amplitude of OAG-induced (291 ± 39 pA, n = 44 cells) vs hypotonicity-induced currents (56 ± 4 pA, n = 84 cells; see above) may also reflect the fact that pharmacological concentrations we used (100–200 μM) were higher than physiologically generated levels of intracellular DAG, resulting in activation of more channels due to production of higher steady-state levels of proximal channel agonists.

In addition to the significant delay observed between application of agonist and the initiation of current, we also found that the rate of NSC current activation by OAG was variable. Although the majority of cells exhibited slowly activating currents such as those shown in Fig. 4,D, in a few cells currents activated very rapidly (see Fig. 4 F). We can only speculate as to the nature of this variability in activation rate using TRPV4 as an example. TRPV4 has been shown to exhibit Ca2+-dependent potentiation mediated by calcium binding to a C-terminal calmodulin binding site (27, 32). It is possible that elevations in intracellular Ca2+ produced by Tg may accelerate the activation kinetics to the extent observed in this example and others like it.

In addition to the metabolic products of DAG such as 5,6-EET, which activates NSCCs in B cells, PKC is a downstream target of DAG that mediates numerous cellular responses, and PKC has been identified as an agonist of certain TRP family NSCCs. Therefore, we also examined the role of PKC in NSCC activation by hypotonicity and OAG. For these experiments, we used a peptide (PKC inhibitory peptide 19-31) that blocks PKC autophosphorylation and protein substrate phosphorylation (33) or the membrane permeant PKC inhibitor RO-31822. Hypotonicity (n = 14 cells) and OAG (n = 7) failed to elicit NSC currents in all cells stimulated in the presence of PKC inhibitors. However, broad spectrum and potent PKC activators, including the phorbol esters phorbol-12,13-didecanoate and PMA, did not activate NSCCs in B cells. Thus, our data suggest that DAG is a mediator of hypotonicity- and pressure-induced NSCCs, but that PKC plays an indirect role in regulating the activity of NSC channels in B cells.

Based upon our observation that hypotonicity activates PLC in B cells and causes Ca2+ release from stores, one would expect mechanical stimuli also to activate store-operated CRAC channels. Surprisingly, although CRAC currents have been recorded in T cells (34), they have not been identified previously in primary B cells. Due to their small conductance (10 fS) (35) and relatively rapid inactivation, CRAC channels would normally be difficult to isolate by patch clamp, and particularly so if activated at the same time as NSC channels, which have a significantly larger conductance. Therefore, we exploited our finding (see above) that PKC inhibitors indirectly block mechanical activation of NSC currents. We found that, in the presence of the PKC inhibitor 19-31, hypotonicity activated a small-amplitude, CRAC-like current that exhibited strong inward rectification and a positive reversal potential (Fig. 5 A).

BCR engagement with anti-IgM F(ab′)2 Ab, which cannot bind to Fc receptors on B cells, also produced a (biphasic) increase in cytoplasmic Ca2+ (Fig. 5,B). Currents produced by BCR engagement, like those elicited with Tg, exhibited inward rectification and a positive reversal potential (Fig. 5,C, top). Moreover, under DVF external conditions, BCR-induced currents were larger in amplitude (5- to 8-fold; Fig. 5 C, bottom) (21) than corresponding currents in Ca2+-containing solution, yet they still exhibited strong inward rectification and a positive reversal potential. However, unlike hypotonic stimulation, which activates distinct CRAC-like and NSC currents, the BCR only activates CRAC-like currents and never elicited an NSC current (n > 50 B cells tested).

Because both the BCR and hypotonicity activate PLCγ-2, we wondered whether the inability of BCR engagement to elicit NSCCs was due to active inhibition or direct regulation by the BCR of NSCC activation or activity. To test this, we first elicited NSC currents with hypotonicity, and then examined the effect of BCR engagement on these currents. We found that the BCR rapidly attenuated hypotonicity-activated NSC currents (Fig. 5 D, n = 10 cells tested), consistent with the idea that BCR-generated signals dynamically regulate NSCC channel activity or activation. A similar effect was observed if B cells were first treated with anti-BCR and then stimulated with hypotonicity (data not shown). These responses to anti-BCR were specific insofar as a mAb against the CD3 component of the T cell Ag receptor did not trigger CRAC currents or suppress the activity of NSCCs in B cells (data not shown).

We next set out to characterize the functional consequences of NSC activation in B cells. In addition to elevating intracellular Ca2+, cation influx subsequent to NSCC activation could alter the membrane potential. To assess this, we used the current clamp mode of the patch clamp to measure the membrane potential of individual B cells. Both hypotonic stress (Fig. 6,A; −80 ± 7 mV to −49 ± 8 mV, n = 12 cells) and hydrostatic pressure (Fig. 6,B; −81 ± 6 mV to −41 ± 4 mV, n = 5 cells) depolarized B cells. OAG induced a larger change from the resting voltage (Fig. 6,C; −75 ± 3.0 mV to −20 ± 5 mV, n = 9), which was consistent with the larger-amplitude NSC currents it elicited. Selective activation of CRAC channels with Tg produced a significantly smaller change (−70.3 ± 3.3 mV to −52.6 ± 3.7 mV, n = 17) in Vm. Low Na+ substitution (Fig. 6, AC) and La3+ (10 μM; Fig. 6,C) each caused a rapid reversal of membrane depolarization consistent with an underlying role for NSC channel-mediated Na+ influx. Moreover, BCR engagement, which attenuates hypotonicity-induced currents (Fig. 5,D), also reversed hypotonicity-induced (data not shown) and OAG-induced depolarization of B cells (Fig. 6 D; −79.7 ± 5.8 mV to −34.3 ± 4.3 mV to 82.0 ± 4.9 mV, n = 3). Thus, BCR engagement attenuates hypotonicity-induced NSCC activity and membrane depolarization resulting from activation of these channels.

FIGURE 6.

Regulation of membrane potential by NSCCs in B cells. Dynamic measurements of Vm recorded using the current clamp mode of the nystatin perforated patch clamp technique. The resting steady-state Vm was obtained from B cells bathed in physiological (155 mM NaCl) external solution and was monitored after stimulation with hypotonicity (A), hydrostatic pressure (B), or OAG (C). Each of these stimuli induced a decrease (depolarization) in steady-state Vm that was reversed by removal of extracellular Na+ (0 mM NaCl, replaced with NMDG Cl). Membrane depolarization induced by OAG (C) and mechanical stimuli (data not shown) were reversed with the channel inhibitor La3+ (100 μM). BCR engagement caused membrane repolarization after initial depolarization induced with OAG (D).

FIGURE 6.

Regulation of membrane potential by NSCCs in B cells. Dynamic measurements of Vm recorded using the current clamp mode of the nystatin perforated patch clamp technique. The resting steady-state Vm was obtained from B cells bathed in physiological (155 mM NaCl) external solution and was monitored after stimulation with hypotonicity (A), hydrostatic pressure (B), or OAG (C). Each of these stimuli induced a decrease (depolarization) in steady-state Vm that was reversed by removal of extracellular Na+ (0 mM NaCl, replaced with NMDG Cl). Membrane depolarization induced by OAG (C) and mechanical stimuli (data not shown) were reversed with the channel inhibitor La3+ (100 μM). BCR engagement caused membrane repolarization after initial depolarization induced with OAG (D).

Close modal

Changes in membrane potential have important consequences for lymphocytes. In T cells, depolarization inhibits cytokine production (36) and activates the β1 integrins (37), which are required for migration and retention of B cells within the spleen (38). However, relatively little is known about the physiological regulation and role of membrane potential in B cells. Given that integrin avidity modulation is important for lymphocyte adhesion and targeting and our data that NSCC activation causes membrane depolarization in B cells, we examined the role of NSC channels in cell binding to the extracellular matrix components ICAM-1 and VCAM-1, which are ligands for the integrins LFA-1 and VLA-4, respectively.

As in previous experiments, OAG and Tg were used to examine the role of NSC and CRAC channels, respectively, in regulating integrin-dependent binding. OAG significantly increased B cell adherence to ICAM-1 (Fig. 7,A) and VCAM-1 (Fig. 7,B), and these increases were blocked by La3+. The La3+ sensitivity of OAG-induced binding suggests that this is an NSC channel-mediated effect of OAG. Importantly, although Tg increases cytoplasmic Ca2+ levels, it did not induce a significant change in Vm (see above) or integrin-dependent binding. Hypotonicity induced a similar increase in ICAM-1 (Fig. 7,C) and VCAM-1 (Fig. 7 D) binding, and these changes (as well as those due to OAG) were sensitive to inhibition by another NSC channel blocker, 2-APB (50 μM). These data represent further evidence that DAG activation of NSCCs mediates mechanical induction of binding. In neither set of experiments did Tg cause any significant increase in integrin binding, suggesting that calcium release and CRAC channel-mediated influx are not sufficient to induce integrin avidity changes.

FIGURE 7.

NSCCs regulate binding of B cells to ICAM-1 and VCAM-1. NSCCs but not CRAC channels regulate binding of B cells to extracellular matrix proteins. Splenic B lymphocytes were stimulated with OAG (200 μM) or Tg (1 μM) to define the role of NSCCs and CRAC channels in cell binding to immobilized ICAM-1 (A) and VCAM-1 (B). OAG induced a significant increase in ICAM-1 (∗, p = 0.04) and VCAM-1 (∗, p = 0.03) binding, which was completely blocked by the channel inhibitor La3+ (ICAM-1: ∗∗, p = 0.05; and VCAM-1: ∗∗, p = 0.02). Tg did not induce an increase in binding to either extracellular matrix component, but it did suppress binding increases produced by OAG (∗∗∗, p = 0.04). Hypoosmotic stimulation of B cells produced an increase in ICAM-1 (C) (∗, p = 0.04) and VCAM-1 binding (D) (∗, p = 0.02) (p < 0.02 for all comparisons) similar to that of OAG (p < 0.01). Induction of binding by hypotonicity and OAG was significantly inhibited (∗∗, p < 0.04) by the channel blocker 2-APB (50 μM). High extracellular K+ (80 mM) also induced a significant increase in ICAM-1 (E) and VCAM-1 (F) binding that was comparable with that induced by hypotonicity and OAG (∗, p ≤ 0.04). BCR engagement (50 μg/ml anti-BCR) itself did not increase binding but did significantly suppress the increases elicited by OAG and hypotonicity (p < 0.05). Each value represents the mean ± SEM for at least three independent experiments. Statistical analysis was performed using Student’s paired t test.

FIGURE 7.

NSCCs regulate binding of B cells to ICAM-1 and VCAM-1. NSCCs but not CRAC channels regulate binding of B cells to extracellular matrix proteins. Splenic B lymphocytes were stimulated with OAG (200 μM) or Tg (1 μM) to define the role of NSCCs and CRAC channels in cell binding to immobilized ICAM-1 (A) and VCAM-1 (B). OAG induced a significant increase in ICAM-1 (∗, p = 0.04) and VCAM-1 (∗, p = 0.03) binding, which was completely blocked by the channel inhibitor La3+ (ICAM-1: ∗∗, p = 0.05; and VCAM-1: ∗∗, p = 0.02). Tg did not induce an increase in binding to either extracellular matrix component, but it did suppress binding increases produced by OAG (∗∗∗, p = 0.04). Hypoosmotic stimulation of B cells produced an increase in ICAM-1 (C) (∗, p = 0.04) and VCAM-1 binding (D) (∗, p = 0.02) (p < 0.02 for all comparisons) similar to that of OAG (p < 0.01). Induction of binding by hypotonicity and OAG was significantly inhibited (∗∗, p < 0.04) by the channel blocker 2-APB (50 μM). High extracellular K+ (80 mM) also induced a significant increase in ICAM-1 (E) and VCAM-1 (F) binding that was comparable with that induced by hypotonicity and OAG (∗, p ≤ 0.04). BCR engagement (50 μg/ml anti-BCR) itself did not increase binding but did significantly suppress the increases elicited by OAG and hypotonicity (p < 0.05). Each value represents the mean ± SEM for at least three independent experiments. Statistical analysis was performed using Student’s paired t test.

Close modal

Because calcium elevations alone are not responsible for changes in integrin binding, then integrin-avidity modulation by NSCC activation could reflect their ability to depolarize the plasma membrane. To test this, we examined the effect of membrane depolarization with high extracellular K+ on integrin-dependent binding. High K+ (80 mM) induced an increase in both ICAM-1 (Fig. 7,E) and VCAM-1 (Fig. 7,F) binding that was similar to increases induced with either OAG or hypotonicity. This response suggests that binding is a direct consequence of NSCC-mediated depolarization of B cells. Given our finding that BCR engagement attenuates hypotonicity-activated NSC currents (Fig. 5,D) and NSSC-mediated depolarization (Fig. 6,D), one would predict that the BCR should similarly attenuate OAG- and hypotonicity-induced binding. As predicted, BCR engagement blocked the increase in B cell binding to ICAM-1 (Fig. 7,E) and VCAM-1 (Fig. 7 F) induced with either hypotonicity or OAG. Together, these data suggest that mechanically activated NSCCs regulate Vm, and thereby the avidity of integrins for extracellular matrix adhesion molecules.

The biophysical properties, pharmacology, and activation mechanisms of NSC currents identified in B cells are similar to a number of TRP family members. Therefore, we screened primary B cells for each of 22 known TRPC, TRPV, and TRPM genes (39). We found that B cells express TRPC2, TRPC3, TRPC6, TRPV2, TRPV4, TRPM1, TRPM5, and TRPM7 mRNA (Fig. 8). The identity of each PCR product was confirmed by DNA sequencing. Furthermore, each primer set was tested on murine cDNA to make certain that negative results reflected an absence of expression and not problems with the primers.

Our efforts to understand mechanisms of Ca2+ signaling in primary B lymphocytes have revealed novel mechanically operated, Ca2+-permeant, nonselective cation channels. These channels are activated by a mechanism that overlaps with proximal steps in BCR-activated pathways (both activate PLC); however, BCR engagement activates only CRAC and not NSCCs. In contrast, hypotonicity activates both NSCCs and CRAC channels. Our data indicate that signals generated by BCR engagement antagonize the activation, but importantly also the functions, of mechanically operated NSCCs. This crosstalk between BCR-operated and mechanically operated signaling pathways represents a novel mechanism by which diverse stimuli can coordinately regulate B cell activation.

We also demonstrate that store-independent NSC and store-operated CRAC-like channels activated in B cells can produce distinct patterns of Ca2+ signaling. Whereas BCR engagement triggers a biphasic change in Ca2+ resulting in a low-amplitude sustained plateau (Fig. 5,B), activation of NSCCs with OAG (Fig. 4,C) or hypotonicity (Fig. 2 A) produces Ca2+ oscillations after the initial spike in concentration. Importantly, the patterns of Ca2+ signaling are similar to those elicited by Ag in naive and tolerized B cells, respectively, that differentially activate transcription factors NFAT and NFκB (40, 41). In previous studies, however, neither the identity of Ca2+ channels nor the signal transduction mechanism responsible for generating distinct patterns of Ca2+ signaling were defined. Although we have not examined anergic cells, our results raise the possibility that patterns of Ca2+ signaling in naive and anergic B cells might reflect regulation in the coupling of the BCR to CRAC and NSCCs in these distinct cell populations.

Our results also indicate that an additional consequence of NSCC (but not CRAC channel) activation in B cells is the activation of integrin-dependent binding. This appears to be independent of the Ca2+ signaling function of NSCCs and related to their ability to regulate the membrane potential. Membrane depolarization has been shown previously to up-regulate MHC class II on B cells (42) and the avidity of integrins expressed on T cells for extracellular matrix components (37). Accordingly, NSCC-mediated changes in Vm alone or in concert with, elevations in Ca2+, may control B cell trafficking within lymphoid compartments, transit to sites of inflammation, docking at key points of primary encounter with blood-borne bacterial pathogens, (e.g., splenic marginal sinuses) interactions with T cells (43). In addition to these immediate responses, mechanical activation of NSCCs by hemodynamic and lymphatic pressure, or membrane strain that results from physical interactions with the extracellular matrix or other cells, could elevate Ca2+ and trigger specific transcriptional or nontranscriptional responses of B cells.

Biophysical and pharmacological properties of currents, including NSCCs we have identified in B cells, are generally not sufficient by themselves for determining their exact molecular identity. In fact, this has not been our goal and is beyond the scope of these current studies. Nonetheless, the biophysical and pharmacological properties of NSC currents recorded in primary B cells point to some candidates to consider. For example, the conductance, Na+:Ca2+ permeability ratio, lack of rectification, La3+ sensitivity, and store-independent, DAG-dependent gating are similar to those of heterologously expressed TRPC3 and TRPC6 (44, 45, 46). In fact, direct interactions between TRPC3 and PLCγ-2 have been observed in DT40 B cells (47), suggesting that a physical interaction may underlie functional coupling between them. Other characteristics of B cell currents, including activation by osmotic stress and by OAG, are consistent with TRV4 channels (27), which are also expressed in B cells (Fig. 8). Heterologously expressed TRPV4 is also activated by hypotonicity in a PKC-independent (phorbol ester-insensitive) manner.

The biophysical properties of NSC currents recorded in primary B cells, however, do not exactly match all those reported for any single TRP family member, raising the possibility that they are encoded by multiple TRP family subunits and the resulting hetero-oligomeric structures possess a hybrid phenotype. Alternatively, it is possible that properties of homomeric channels expressed heterologously in cell lines differ from those of identical channels expressed in primary B cells. A factor as elementary as the expression level of TRPC3 dictates whether it is activated in an (IP3) store-dependent or store-independent manner (48). Thus, discrepancies that we observe could reflect differences associated with physiological expression of channels in primary B cells vs cell lines. It is also possible that NSCCs we have identified are not TRP family members or members of any known group of channels, although this is considered to be unlikely based upon numerous similarities we have identified between currents and expressed TRP family members.

The most direct way to determine which of any TRP family members are responsible for currents we have identified would be to use genetic methods to alter channel expression. Because primary murine B cells are not amenable to transfection, simple methods cannot be used to do this, and while such manipulations could be tested in DT40, A40, or Ramos B cell lines, these comparisons have drawbacks, including the fact that transformed cell lines do not exactly recapitulate responses of primary mammalian cells. Consequently, efforts are underway that use more complex genetic strategies to directly examine the roles of TRPC3, TRPC6, and TRPV4 in mechanical signaling responses of primary B cells in vivo.

TRP channels have been implicated in the sensory responses of cells to environmental factors, including osmolarity, pressure, heat, cold, oxidative stress, phermones, hormones, and phospholipids, and their activation is regulated by a variety of intracellular phospholipid products, including DAG, phosphatidylinositol-4,5-bisphosphate, arachadonic acid, epoxyeicosanoids, and others downstream from PLC (7). Although we have used hypotonicity to generate mechanical strain in the membrane, the experimental extremes in tonicity that we have used may not be encountered in vivo under physiologically relevant conditions. We propose that these responses to hypotonicity and hydrostatic pressure represent a generalized signaling response activated in B lymphocytes by mechanical or physical stress. Although NSCCs, including TRPV4, have well-documented sensitivity to cell volume changes and TRPV4 channels, has been implicated in mechano-sensing (reviewed in Ref. 49) by a DAG-dependent process, we have not specifically examined the role of NSCCs in volume regulation. We speculate that cation fluxes through these channels upon activation by hypotonicity in vivo could play a role, along with K+ and Cl channels, in regulating the volume and viability of B cells. Mechanically induced changes in cell volume may also help B cells during transit from the vasculature to, or within, sites of inflammation. In monocytes, mechanical stress also produces PLC-dependent intracellular Ca2+ elevations and increases integrin-dependent cell adhesion (50).

In summary, our results are the first documentation of a link between PLC activity and mechanical stimulation, between PLC and TRP channel activation, and between TRP channel activation and integrin functions in primary B cells. Our data demonstrate that PLCγ-2 integrates the responses of B cells to Ag receptor and mechanical stimuli with two major physiological consequences. First, NSCCs play an important role in regulating the membrane potential of B cells and in activating surface integrins. Second, NSCCs in B cells are also Ca2+ permeant and their activation results in oscillatory changes in the concentration of cytoplasmic Ca2+.

The bias toward NSCC or CRAC channel activation by BCR and innate stimuli could, therefore, represent a novel mechanism by which these two Ca2+ channels produce distinct patterns of Ca2+ signaling and specify distinct Ca2+-dependent responses of B cells. Because CRAC channel activation is associated with biphasic changes and NSCC activation with oscillations in Ca2+, distinct channels may underlie the preferential activation of transcription factors such as NFAT or NF-κB, which are capable of discriminating between these different Ca2+ signals. Selective activation of CRAC channels by BCR engagement may be responsible for biphasic changes, which preferentially activate NFAT, whereas sustained low-frequency Ca2+ oscillations produced by hypotonicity and DAG (Figs. 2 and 4), previously shown to activate NFκB (40), may reflect the dominant activity of NSCCs (see model, Fig. 9). Interestingly, shear-induced, Ca2+-regulated NFκB activation has been described in several other cell types, including neutrophils (51) and osteoblasts (52). Although NSCCs are not activated by BCR engagement in naive B cells, it is possible that costimulatory or coinhibitory receptors may differentially regulate the activity of NSCC and CRAC channels and thereby produce distinct physiological and immunological fates in them. Moreover, Ca2+ oscillations produced by Ag in anergic B cells (53) may be due to differences in the coupling between the BCR and NSCCs in this population.

FIGURE 9.

A revised model of Ca2+ signaling proposed for B cells. Mechanical stimuli (MR, mechanical receptor) activate CRAC and NSCCs (yellow) in B cells, whereas BCR engagement activates CRAC, but not NSCCs. Activation of PLCγ-2 by mechanical stimuli catalyzes the formation of IP3 and diacylglycerol from membrane phosphatidylinositol-4,5-bisphosphate. IP3 binds to IP3 receptor/channels on intracellular (endoplasmic reticulum (ER)) stores and triggers the release of Ca2+ from these stores. CRAC channels are subsequently activated by intracellular Ca2+ store depletion, and Ca2+ influx produces a sustained elevation in Ca2+ concentration. DAG produced by PLCγ-2 activates PKC and NSCCs. Extracellular Ca2+ and Na+ entry through NSCCs produce changes in cytoplasmic Ca2+, but they also depolarize the plasma membrane. Depolarization triggers an increase in integrin avidity for extracellular matrix components. Previous studies have demonstrated that steady-state elevations in Ca2+, such as those produced by BCR engagement, activate NFAT and NFκB and reflect the dominant activity of CRAC channels. Sustained low-frequency Ca2+ oscillations, such as those produced by NSCCs have previously been shown to preferentially activate the DAG/PKC-regulated transcription factor NFκB (41 ).

FIGURE 9.

A revised model of Ca2+ signaling proposed for B cells. Mechanical stimuli (MR, mechanical receptor) activate CRAC and NSCCs (yellow) in B cells, whereas BCR engagement activates CRAC, but not NSCCs. Activation of PLCγ-2 by mechanical stimuli catalyzes the formation of IP3 and diacylglycerol from membrane phosphatidylinositol-4,5-bisphosphate. IP3 binds to IP3 receptor/channels on intracellular (endoplasmic reticulum (ER)) stores and triggers the release of Ca2+ from these stores. CRAC channels are subsequently activated by intracellular Ca2+ store depletion, and Ca2+ influx produces a sustained elevation in Ca2+ concentration. DAG produced by PLCγ-2 activates PKC and NSCCs. Extracellular Ca2+ and Na+ entry through NSCCs produce changes in cytoplasmic Ca2+, but they also depolarize the plasma membrane. Depolarization triggers an increase in integrin avidity for extracellular matrix components. Previous studies have demonstrated that steady-state elevations in Ca2+, such as those produced by BCR engagement, activate NFAT and NFκB and reflect the dominant activity of CRAC channels. Sustained low-frequency Ca2+ oscillations, such as those produced by NSCCs have previously been shown to preferentially activate the DAG/PKC-regulated transcription factor NFκB (41 ).

Close modal

Regardless of the physiological function of NSCCs, the observed overlap in proximal mechanisms of NSCC and CRAC channel activation that we have identified (Fig. 3), and the dynamic regulation of NSCC activity and function by BCR-generated signals (Figs. 5 and 8), have broad implications for the physiological mechanisms, but also pharmacological regulation, of B cell Ca2+ signaling, integrin activation, and immune responses.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

Abbreviations used in this paper: IP3, inositol 1,4,5-triphosphate; CRAC, Ca2+-release-activated calcium; NSCC, nonselective cation channel; TRP, transient receptor potential; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; Tg, thapsigargin; NMDG, N-methyl-d-glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; Vm, membrane voltage potential; OAG, 1-oleoyl-2-acetyl-sn-glycerol; mOsm, milliosmolar; 2-APB, 2-aminoethyldiphenyl borate; DVF, divalentcation free; 5,6-EET, 5,6-epoxyeicosa-8Z,11Z,14Z-trienoic acid.

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