316 GPR55-(in)dependent ion signalling of LPI A Bondarenko et al Figure 6 LPI-evoked membrane currents that account for mem- brane depolarization. Representative tracings of membrane currents induced by 5 mM LPI measured at holding potentials of -75 and +75 mV (n = 7). In the whole-cell mode, 1 s voltage ramps from -80 mV to +80 mV were applied every 5 s from a holding potential of -40 mV (A). Current-voltage relationship constructed from current responses to voltage ramps before and after application of 5 mM LPI at points speciﬁed in A (B). Statistical evaluation of the effects of 5 mM LPI on endothelial cell membrane potential under control conditions (n = 7) and in the presence of 100 mM DIDS (n = 3) (C). GPR55. This conclusion is based on our ﬁndings that the antagonist of GPR55, rimonabant (Lauckner et al., 2008; Waldeck-Weiermair et al., 2008) and siRNA-mediated GPR55 knock-down prevented the rapid intracellular Ca2+ release in response to LPI, conﬁrming our earlier results that LPI trig- gered intracellular Ca2+ mobilization via GPR55 (Waldeck- Weiermair et al., 2008). Moreover, our ﬁndings that the inhibition/knock-down of GPR55 prevented LPI-induced transient membrane hyperpolarization and outward current while GPR55 over-expression strongly enhanced LPI-induced hyperpolarization, further supports our assumption that the initial GPR55-mediated Ca2+ elevation/release is accompanied by activation of plasma membrane ion channels, that were characterized as intermediate and large conductance Ca2+- activated K+ channels, the main carrier for Ca2+-induced mem- brane hyperpolarization in this cell type (Frieden and Graier, British Journal of Pharmacology (2010) 161 308–320 2000; Malli et al., 2003). In contrast to other G protein- coupled receptor agonists in this cell type (Malli et al., 2007), LPI initiated a transient Ca2+ elevation even in the presence of extracellular Ca2+, which points to a limited activity of store- operated Ca2+ entry (SOCE). Nevertheless, LPI also induced inward currents that were not linked to increased cytosolic Ca2+. These ﬁndings were in apparent contradiction to reports describing LPI as direct activator of SOCE in smooth muscle cells (Smani et al., 2003; 2007; Singaravelu et al., 2006) and the hypothesis that LPI represents a Ca2+-independent, phos- pholipase A2-derived, mediator for activation of SOCE (Smani et al., 2004). These differences might be due to the expression of a wide variety of Ca2+-permeable ion channels in different cell types. Moreover, because LPI-triggered sustained mem- brane depolarization and inward current were not affected by inhibition of GPR55 by rimonabant or the molecular manipu- lation of GPR55 expression, LPI obviously exhibited its sus- tained effects independently of this orphan receptor. This assumption was further supported by our ﬁndings that LPI- induced slow cellular Na+ loading was independent of the expression level of GPR55. Accordingly, the LPI-induced biphasic responses of the membrane potential differs from the reported monophasic hyperpolarization elicited by LPC in endothelial cells (Erdogan et al., 2007) and coronary artery smooth muscle cells (Terasawa et al., 2002), which respond by a slowly devel- oping depolarization due to activation of a non-selective cation current. However, the biphasic effect of LPI on electri- cal responses reported here resembles the effect of LPC on renal arterial smooth muscle cells (Jabr et al., 2000). To investigate the nature of the GPR55-independent inward current, blockers of several endothelial transporters and chan- nels were tested. As LPI-evoked depolarization was not affected by the Cl- channel blocker DIDS, the involvement of Cl- channels (Nilius and Droogmans, 2001) in the membrane depolarization to LPI is unlikely. In contrast, depletion of Ca2+ in the bath solution, which produced gradual depolarization possibly due to inhibition of basal Ca2+ entry and a subsequent reduction of BKCa channel activity, strongly reduced LPI-induced depolarization. More- over, the Ca2+ entry blocker Ni2+ produced a gradual depolar- ization and diminished LPI-induced depolarization, thus, indicating that LPI-induced sustained depolarization requires Ca2+ entry. We also used La3+, which selectively inhibits SOCE and Ca2+ permeable non-selective cationic channels (Nilius and Droogmans, 2001; Terasawa et al., 2002) at low concen- trations. Our ﬁndings that La3+ attenuated the LPI-induced depolarization further support our assumption of the impor- tance of Ca2+ entry for LPI-induced depolarization and point to a GPR55-independent activation of Ca2+ permeable non- selective cationic channel(s) by LPI. Our experiments further showed that LPI increased also the Na+ permeability of endothelial cells. After cell dialysis with Cs+- containing solution, LPI evoked a linear current that reversed at 0 mV, a property of non-selective cationic current (Jabr et al., 2000; Terasawa et al., 2002). Na+ substitution with choline inhibited the current, indicating a signiﬁcant Na+ permeability stimulated by LPI, which contributed to mem- brane depolarization. Furthermore, a direct slow and GPR55- independent Na+ loading was observed in response to LPI,