312 GPR55-(in)dependent ion signalling of LPI A Bondarenko et al Figure 2 The GPR55 blocker rimonabant prevented only the initial Ca2+ transients and membrane hyperpolarization evoked by LPI. Cells were pre-treated with 1 mM rimonabant as shown. This GPR55 blocker abolished the transient elevation of the cytosolic-free Ca2+ concentration in response to 5 mM LPI (A). Typical membrane potential recording showing the inhibitory effect of rimonabant (1 mM) of a LPI (5 mM)-evoked hyperpolarization (B). Statistical evaluation of LPI (5 mM)-induced biphasic changes in endothelial cell membrane potential under control conditions (LPI, n = 9) and in the presence of 1 mM rimonabant (n = 14) (C). Rimonabant (1 mM) prevented the development of the initial outward current (1st phase) but failed to prevent the sustained inward current (2nd phase) in response to 5 mM LPI (n = 3) (D). *P < 0.005 versus control. These data indicate that the initial and transient ionic responses to LPI essentially depend on GPR55, while the sub- sequent, slowly developing, sustained electrical responses appear to be independent of this orphan receptor and medi- ated via direct effects on non-selective cation channels in endothelial cells. LPI-induced changes in membrane potential are due to different currents To describe the transmembrane currents that underlie the LPI-induced changes in membrane potential, experiments in the voltage clamp mode were performed. When membrane voltage was kept at -40 mV, which is close to the resting membrane potential (-40.9 2.2 mV; n = 23) of this cell type, 5 mM LPI induced a biphasic response that showed a transient outward current (Hyperpolarization phase) followed by a sus- tained inward current (Depolarization phase) (EB#1, PS#1) (Figures 1G and 5A). Hyperpolarization phase. Voltage ramps between -80 to +80 mV during the hyperpolarization (outward current) phase (Figure 5A) revealed a current potential curve with the common characteristics of an agonist-activated current, mainly achieved by BKCa channels in whole cell recordings of these endothelial cells (Figure 5B) (Frieden and Graier, 2000). In agreement with this assumption, LPI induced rapid activa- tion of large conductance channels in the cell-attached con- ﬁguration (Figure 5C). In the whole-cell conﬁguration, the outward current did not appear when pipettes were ﬁlled with British Journal of Pharmacology (2010) 161 308–320 Ca2+-free Cs+-containing solution (PS#2) in order to avoid KCa currents (data not shown). Moreover, membrane hyperpolar- ization to LPI was sensitive to charybdotoxin (Figure 5D). Charybdotoxin (100 nM) produced a gradual endothelial depolarization (Figure 5D) that points to the contribution of charybdotoxin-sensitive KCa channels to the resting mem- brane potential (Ledoux et al., 2008). Altogether, these results indicate that the LPI-induced GPR55-dependent hyperpolar- ization is due to an activation of large conductance, Ca2+- dependent, charybdotoxin-sensitive, K+ channels. Depolarization phase. Next, the sustained inward current that accounts for the slowly developing membrane depolarization in endothelial cells in response to LPI was investigated. Using voltage ramps between -80 to +80 mV in the inward current phase (Figure 6A) revealed a linear current-potential curve (Figure 6B). We intended to characterize the nature of the current(s) that was/were responsible for the LPI-induced membrane depolarization in endothelial cells. First, the involvement of Cl- channels in LPI-induced depolarization was tested using the Cl- channel blocker 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). DIDS (100 mM and 1 mM) failed to prevent endothelial depolariza- tion (Figure 6C) evoked by 5 mM LPI, thus, suggesting that Cl- channels are unlikely to be involved in the LPI-induced elec- trical responses in endothelial cells. To evaluate the role of Ca2+ entry in the LPI-induced elec- trical responses, LPI was added either in nominal Ca2+-free or Ni2+-containing solution. Ca2+ withdrawal evoked a gradual depolarization with the amplitude of 11.4 2.1 mV (n = 6). In