The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca2+ -activated K+ channels

Abstract

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca(2+) and voltage-gated potassium (BK(Ca)) channels. Here, we explored the effect of LPI on intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels using excised inside-out patches from endothelial cells. The functional expression of IK(Ca) was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK(Ca) channels with a slope conductance of 39 pS in symmetric K(+) gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3-10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK(Ca) single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK(Ca) single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.

Fig. 1

Effect of IK_Ca channel inhibitors on endothelial hyperpolarization. a Typical membrane potential recording showing the effect of charybdotoxin (100 nM) applied during the plateau phase of hyperpolarization to 10 μM histamine. b Typical membrane potential recording showing the effect of TRAM-34 (2 μM) applied during the plateau phase of hyperpolarization to 10 μM histamine. c Typical membrane potential recording showing the effect of iberiotoxin (200 nM) applied during the plateau phase of hyperpolarization to 10 μM histamine. d Typical membrane potential recording showing the effect of TRAM-34 (2 μM) applied during the plateau phase of hyperpolarization to 10 μM ATP. e Typical membrane potential recording showing the effect of preexposure to charybdotoxin (100 nM) on hyperpolarization to 10 μM histamine. f Typical membrane potential recording showing the effect of preexposure to TRAM-34 (2 μM) on hyperpolarization to 10 μM histamine. g Typical membrane potential recording showing the effect of preexposure to iberiotoxin (200 nM) on hyperpolarization to 10 μM histamine. h Statistical representation of the effect of charybdotoxin (100 nM; n = 4), TRAM-34 (2 μM; n = 5) and iberiotoxin (200 nM; n = 5) on peak hyperpolarization to 10 μM histamine (n = 11). *P < 0.05 vs control hyperpolarization

Fig. 2

Functional IK_Ca channels are present in excised patches from EA.hy926 cells. a Representative single-channel recording out of seven independent experiments in inside-out patch exposed to 1 μM bath Ca²⁺ showing two types (large and intermediate conductance) of Ca²⁺-activated K⁺ single-channel activity. b Representative single-channel recording from four independent experiments in inside-out patch in the presence of bath Ca²⁺ as indicated. The patch was held at a holding potential of −80 mV. Pipette solution contained iberiotoxin (400 nM) and apamin (100 nM). Single-channel activity is denoted as downward deflections. Bars from the right indicate closed state of the channel

Fig. 3

Voltage dependency of the IK_Ca channel. a Representative recordings out of three independent experiments showing the single IK_Ca channel activity at different voltages in patch containing one active channel in outside-out configuration in symmetrical K⁺ and free [Ca²⁺] in the pipette 10 μM. Bars from the left indicate the closed state of the channel. b Voltage dependency of Po presented in a. c Current–voltage relationship of IK_Ca in symmetrical K⁺ condition

Fig. 4

Determination of the CMC value for LPI. a and b Determination of CMC level using capillary height method. LPI-evoked changes in capillary height normalized to the mean capillary height level attained in LPI-free 140 mM K⁺-containing bath solution presented in linear (a) and log scale (b) to expand for clarity the data points derived from high and low LPI concentrations, respectively. Arrows indicate the point where the curve plateaued corresponding to the CMC level. The number of individual measurements is indicated above the averaged data points. c Determination of CMC level for LPI using the fluorescence method. LPI was added cumulatively to a solution containing 0.25 μM dephenylhexatriene in 140 mM K⁺-based bath solution and the rise in fluorescence at 30 μM LPI corresponds to transition to micelle aggregation. The excitation was 345 nm, fluorescence emission was 430 nm. Representative record out of five experiments

Fig. 5

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 1 and 3 μM LPI in the presence of 1 μM Ca²⁺ in the bath at a holding potential of −80 mV. Channel openings are shown as downward deflections. b Representative single-channel recording in inside-out patch exposed to 1 μM Ca²⁺ showing concentration-dependent activation of IK_Ca channel activity in response to bath application of 1, 3, and 10 μM LPI, which was reversible following 15 min of wash out of LPI. Channel openings are shown as downward deflections and bars from the left indicate the zero current level. c Statistical representation of the effect of 0.3 (n = 4), 1 (n = 5), 3 (n = 14), and 10 μM (n = 3) LPI on IK_Ca channel activity in the presence of 1 μM Ca²⁺ at a holding potential of −80 mV. *P < 0.05 vs basal NPo in the absence of LPI. d A representative amplitude histogram before (Control) and after exposure of excised patch to 3 μM LPI in the presence of 1 μM Ca²⁺. Data points were fitted by Gaussian fits. e Statistical representation of the effect of 3 μM LPI on IK_Ca channel activity in the presence of 10 μM (n = 4) and 1 μM Ca²⁺ (n = 14) at a holding potential of −80 mV

Fig. 6

Effect of LPI on the kinetic properties of IK_Ca channels. a Representative single-channel recording in inside-out patch containing one active channel exposed to 1 μM Ca²⁺ prior (Control) and during the exposure to 1 μM LPI at the cytosolic side of the patch at a holding potential of −80 mV. Channel openings are shown as downward deflections. b The mean open time of the IK_Ca prior (Control) and after addition of 3 μM LPI (n = 9). c The mean closed time (frequency of openings) of the IK_Ca prior (Control) and after addition of LPI (n = 9). *P < 0.05 vs. control. d Open time histograms in the absence (Control) and presence of 1 μM LPI. In both cases, the open time was fitted to two exponentials (τo1 and τo2). LPI did not affect τo1 and slightly increased τo2. e Closed time histograms in absence (Control) and presence of 1 μM LPI. In both cases, the closed time was fitted to three exponentials (τc1, τc2, and τo2). LPI did not affect τc1, slightly decreased τc2, and strongly decreased τc3

Fig. 7

Intracellular LPI potentiates endothelial cell hyperpolarization to histamine via activation of IK_Ca channels. a Statistical analysis of the effect of 2 μM TRAM-34 on histamine- (0.5 μM) induced hyperpolarization in the absence (control, n = 12; TRAM-34, n = 5) or presence of 1 μM LPI in the pipette (control, n = 14; TRAM-34, n = 4). b Representative traces in membrane potential out of 14 independent experiments showing endothelial cell hyperpolarization to bath application of 0.5 μM histamine in the presence of 1 μM LPI in the patch pipette and its sensitivity to TRAM-34 (2 μM)