The GPR55 agonist lysophosphatidylinositol acts as an intracellular messenger and bidirectionally modulates Ca2+ -activated large-conductance K+ channels in endothelial cells

Abstract

Lysophospholipids are known to serve as intra- and extracellular messengers affecting many physiological processes. Lysophosphatidylinositol (LPI), which is produced in endothelial cells, acts as an endogenous agonist of the orphan receptor, G protein-coupled receptor 55 (GPR55). Stimulation of GPR55 by LPI evokes an intracellular Ca(2+) rise in several cell types including endothelial cells. In this study, we investigated additional direct, receptor-independent effects of LPI on endothelial large-conductance Ca(2+) and voltage-gated potassium (BK(Ca)) channels. Electrophysiological experiments in the inside-out configuration revealed that LPI directly affects the BK(Ca) channel gating properties. This effect of LPI strictly depended on the presence of Ca(2+) and was concentration-dependent, reversible, and dual in nature. The modulating effects of LPI on endothelial BK(Ca) channels correlated with their initial open probability (Po): stimulation at low Po (<0.3) and inhibition at high Po levels (>0.3). In the whole-cell configuration, LPI in the pipette facilitated membrane hyperpolarization in response to low (0.1-2 μM) histamine concentrations. In contrast, LPI counteracted membrane hyperpolarization in response to supramaximal cell stimulation with histamine. These results highlight a novel receptor-independent and direct bidirectional modulation of BK(Ca) channels by LPI on endothelial cells. We conclude that LPI via this mechanism serves as an important modulator of endothelial electrical responses to cell stimulation.

Fig. 1

Ca²⁺ is required for the direct modulation of BK channel activity by LPI. a Representative single-channel recordings in inside-out patch (patch potential = +40 mV) exposed to 0.1 (top panels) and 0.3 μM (middle panels) Ca²⁺ prior (control) and after addition of 3 μM LPI to the cytosolic side of the patch. Channel openings are shown as upward deflections (c, closed, o, open). In the absence of Ca²⁺, LPI has no effect on BK_Ca channel activity (lower panels). b Summary data for the effect of 3 μM LPI on BK_Ca channel activity in the absence (0 mM Ca²⁺; n = 6) and presence of 0.1 (n = 10) and 0.3 μM (n = 21) free Ca²⁺ in the bath. *p < 0.05 vs. basal NPo in the absence of LPI.

Fig. 2

The LPI effect develops slowly, is sustained, reversible, and occurs in a concentration-dependent manner. a Representative single-channel recording in inside-out patch exposed to 10 μM LPI in the presence of 0.3 μM bath Ca²⁺ holding potential (Vm) = −40 mV. Channel openings are shown as downward deflections. b The reversibility of the effect of LPI effect is demonstrated by this representative single-channel recording in inside-out patch exposed to 0.3 μM Ca²⁺ prior (top, control), during (middle), and 15 min after exposure to 10 mM LPI (Vm = −40 mV). c Representative single-channel recording in inside-out patch exposed to 0.3 μM Ca²⁺ showing concentration-dependent activation of BK_Ca channel activity in response to bath application of 1, 3, and 10 μM LPI. Channel openings are shown as downward deflections (c, closed, o, open state). d Statistical representation of the effect of 1 (n = 6), 3 (n = 7), and 10 μM (n = 4) LPI on BK_Ca channels in the presence of 0.3 μM Ca²⁺ at Vm = −40 mV. *p < 0.05 vs. basal NPo in the absence of LPI

Fig. 3

LPI-evoked increase in BK_Ca channel activity is mainly due to a marked decrease in mean closed time. a Representative single-channel recording in inside-out patch containing one active channel exposed to 0.3 μM Ca²⁺ prior (control) and during exposure to 3 μM LPI at the cytosolic side of the patch (Vm = 40 mV). Channel openings are shown as upward deflections (c, closed, o, open states). b The mean open time of the BK_Ca prior (control) and after addition of 3 μM LPI (n = 12). c The mean closed time (frequency of openings) of the BK_Ca prior (control) and after addition of LPI (n = 12). *p < 0.05 vs. control

Fig. 4

The dual effect of LPI on BK_Ca channel activity. a Effect of 3 μM LPI on BK_Ca channel activity at different bath Ca²⁺ concentrations that correspond to basal (0.1 μM; n = 10), moderately (0.3 μM; n = 21), strong (1 μM; n = 20), and maximally (10 μM; n = 20) elevated cytosolic Ca²⁺ levels. *p < 0.05 vs. basal NPo in the absence of LPI. b Representative single-channel recordings from multichannel patch exposed to 1 μM Ca²⁺ at Vm = +40 mV with high basal NPo showing inhibitory effect of 3 μM LPI on BK_Ca channel activity. Channel openings are shown as upward deflections (c, closed; o, open state). Right panel: statistical representation of Po values before (control) and after addition of 3 μM LPI in patches exposed to 1 μM Ca²⁺ at Vm = ±40 mV and responded by a decrease in BK_Ca channel activity. c Representative single-channel recording in inside-out patch containing one active channel with the basal Po = 0.57 showing inhibitory effect of 3 μM LPI on BK_Ca channel activity. The patch was exposed to 1 μM Ca²⁺ at Vm = +40 mV. d Summary data of the concentration-dependency of the inhibitory effect of LPI on BK_Ca channel activity in patches exposed to 1 μM Ca²⁺ at Vm = +40 mV (n = 6–21) *p < 0.05 vs. control. The respective mean open time (e; n = 6, *p < 0.05) and the mean closed time (f; n = 6, *p < 0.05) of the BK_Ca channel prior (control) and after addition of 3 μM LPI (Vm = +40 to +60 mV). Data collected from patches exposed to 1 and 10 μM Ca²⁺ were pooled. g Correlation between the LPI-evoked alterations in BK_Ca channel activity (expressed as the ratio of NPo values in the presence and absence of 3 μM LPI) and basal Po values at Vm = +40 mV. Data points were obtained in the presence of 0.1, 0.3, 1, and 10 μM Ca²⁺

Fig. 5

Effect of LPI on voltage-sensitivity of BK_Ca channel. a The activity of BK_Ca channels prior (left) and after (right) bath application of 3 μM LPI. The patch was exposed to 1 μM Ca²⁺. Unitary currents were recorded at different membrane potentials as indicated. Upward deflections are the opening events of the channel (c, closed, o, open state). b Correlation of the effect of 3 μM LPI on the channels Po expressed as percent of control with the actual holding potential. The relationship between the mean open time (c) and mean closed time (d) of the channel with the actual holding potential in the absence (control) and presence of LPI (3 μM). Data presented are representative data and experiments were repeated four times with different patches that provided similar results

Fig. 6

The dual effect of LPI depends on the level of basal BK_Ca channel activity. a Representative single-channel recording in inside-out patch exposed to a single Ca²⁺ concentration of 10 μM at different voltages. LPI (3 μM) increases NPo at negative voltages (Vm = −40 and Vm = −60 mV) and decreases NPo at positive voltages (Vm = +40 and Vm = +60 mV), where NPo is high (c, closed, o, open state). b Graphical representation of the dual effect of LPI at different voltages in the same patch. c Graphical representation showing Po values in the absence (control) and presence of 3 μM LPI at different voltages in the same patch

Fig. 7

Intracellular LPI potentiates endothelial cell hyperpolarization to low histamine concentrations through BK_Ca channels. a, b, c Representative endothelial cell hyperpolarization to bath application of 0.1, 0.5, and 2 μM histamine under control conditions (no LPI in patch pipette; a), or in the presence of 0.1 (b) and 1 μM LPI (c) in patch pipette. d Statistical representation of the hyperpolarizing effect of various moderate histamine concentrations (0.1, 0.5, and 2 μM) in the absence or presence of 0.1 or 1 μM LPI into patch pipette [No LPI, 0.1 (n = 19), 0.5 (n = 16), and 2 μM histamine (n = 7); 0.1 μM LPI, 0.1 (n = 10), 0.5 (n = 10), and 2 μM histamine (n = 7); 1 μM LPI, 0.1 (n = 6), 0.5 (n = 7), and 2 μM histamine (n = 5)]. *p < 0.05 vs. the absence of LPI in the pipette. e Representative membrane potential recording showing inhibitory effect of iberiotoxin (100 nM) on potentiated by intrapipette LPI (1 μM) endothelial cell hyperpolarization to 2 μM histamine. f Statistical representation of the effect of iberiotoxin (100 nM) on endothelial cell peak hyperpolarization to 0.5 and 2 μM histamine with and without 1 μM LPI in the pipette; (histamine 0.5 μM − no LPI in pipette ± iberiotoxin, n = 8; 1 μM LPI in pipette ± iberiotoxin, n = 7; histamine 2 μM − no LPI in pipette ± iberiotoxin, n = 8, 1 μM LPI in pipette ± iberiotoxin, n = 11)

Fig. 8

Intracellular LPI counteracts endothelial cell hyperpolarization to supramaximal histamine concentrations. a–c Representative endothelial cell hyperpolarization to bath application of 100 μM histamine under control condition (a) and under condition of cell dialysis with 1 μM LPI via patch pipette in the absence (b) and presence (c) of external iberiotoxin (IbTx, 100 nM). d Statistical representation of the effect of cell dialysis with 1 μM LPI in the absence and presence of external 100 nM iberiotoxin (IbTx) on peak endothelial cell hyperpolarization to supramaximal histamine concentration (i.e., 100 μM). *p < 0.1 vs. in the absence of LPI in the pipette and IbTx in the bath (control, n = 24; LPI, n = 40; IbTx + LPI, n = 9). e Statistical representation of the effect of cell dialysis with 1 μM LPI in the absence and presence of external 100 nM iberiotoxin (IbTx) on sustained endothelial cell hyperpolarization to supramaximal histamine concentration (i.e., 100 μM). Results are expressed as the ratio of mean membrane potential values at 300th second after the peak and at the peak of hyperpolarization. **p < 0.05 vs. in the absence of LPI in the pipette and IbTx in the bath (control, n = 24; LPI, n = 40; IbTx + LPI, n = 9)