Volume 11, Issue 2, Pages 121-124 (January 2001) Gα13 mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells  Friso R. Postma, Kees Jalink, Trudi Hengeveld, Stefan Offermanns, Wouter H. Moolenaar  Current Biology  Volume 11, Issue 2, Pages 121-124 (January 2001) DOI: 10.1016/S0960-9822(01)00030-6

Figure 1 (a) LPA (1 μM) evokes a slowly developing, prolonged membrane depolarization in serum-starved (24 hr) N1E-115 cells, which is associated with an increase in membrane conductance (decrease in membrane resistance) as measured by voltage responses to brief hyperpolarizing current pulses. The initial transient hyperpolarization (time zero) is due to a Ca2+-activated K+ conductance. Peak depolarization = −17 ± 2.0 mV (means ± SEM, n = 17). A second addition of LPA is shown to have no effect. An identical response was observed in NG108-15 cells. Intracellular microelectrode recordings were performed as described [2, 3]. Depolarization was accompanied by rapid RhoA-mediated growth cone collapse and neurite retraction. (b–d) Inward currents in N1E-115 cells induced by LPA (1 μM), S1P (1 μM), and thrombin receptor activating peptide (TRP, 100 μM), as measured by the amphotericin-perforated patch-clamp technique [3]. Holding potential: −60 mV. (e) Bradykinin (BK, 1 μM), which mobilizes Ca2+ but fails to activate RhoA in N1E-115 cells, elicits a small outward Ca2+-activated K+ current followed by a small inward current (a mixed cation current, carried by both Na+ and K+; F. R. P., unpublished data) Current Biology 2001 11, 121-124DOI: (10.1016/S0960-9822(01)00030-6)

Figure 2 Cl−-mediated membrane depolarization, in common with RhoA-mediated neurite retraction, is independent of phospholipase C (PLC) and Ca2+ mobilization. (a) Dissection of PLC activity from depolarization and neurite retraction. Cells were stimulated with LPA, S1P, or bradykinin (BK, 1 μM), as indicated. PLC activity was assayed by measuring the production of total inositol phosphates, as described [21]. A double plus sign denotes full contractile or electrophysiological responses; a hyphen denotes no response. (b) Typical Ca2+ responses to various GPCR agonists and ionomycin (1 μM), as recorded in a single N1E-115 cell by the use of fluorescence ratio recording. Note that S1P fails to evoke a Ca2+ signal, yet it is a potent inducer of Cl−-mediated membrane depolarization and RhoA-mediated neurite retraction. Conversely, bradykinin (BK) elicits a Ca2+ signal without inducing depolarization and neurite retraction. Serum-starved N1E-115 or NG108-15 cells were loaded for 15 min with a 1:10 mixture of Calcium-Green-AM (5 μM) and Fura-Red-AM in culture medium. Experiments were performed at 37°C on a Zeiss Axiovert microscope in bicarbonate-buffered saline. Excitation was at 485 ± 5 nm, and emissions were measured with two photomultipliers equipped with a bandpass filter 500–560 nm (green signal) and a 590 nm long-pass filter (red signal). Data collection and ratio analysis was carried out with FELIX software (Photon Technology International) Current Biology 2001 11, 121-124DOI: (10.1016/S0960-9822(01)00030-6)

Figure 3 Involvement of Gα13. (a) Injection of antibodies against Gα12 in N1E-115 cells was without effect on the inward current (n = 18), as was microinjected rabbit serum (diluted 1:1) and anti-Gαi antiserum (not shown). Control experiments revealed that the anti-Gα12 antibody does inhibit agonist-induced RhoA activation, as evidenced by impaired f-actin assembly (not shown). (b) In anti-Gα13-injected cells, LPA (1 μM) and thrombin receptor-activating peptide (TRP, 100 μM) fail to activate the inward current, whereas the outward current (through Ca2+-activated K+ channels) remains unaffected (n = 16). Membrane conductance was monitored by the application of brief voltage steps (10 mV, 200 ms). Holding potential = −60 mV. Results were tested for statistical significance with a binomial test. No group differed significantly from control-injected cells except for the cells injected with anti-Gα13 (p < 0.005; n = 16). (c) LPA-induced inward current in control embryonic fibroblasts. The figure shows representative recording of at least 10 different experiments. (d) Lack of response to LPA in Gα13−/− fibroblasts. Representative recording of 10 different experiments. (e) Transfection of wild-type Gα13 into Gα13−/− cells restores the LPA-induced inward current (n = 4). (f) Transfection of the inactive mutant Gα13G225A [14] fails to restore the response to LPA (n = 4) Current Biology 2001 11, 121-124DOI: (10.1016/S0960-9822(01)00030-6)

Figure 4 Membrane depolarization induced by LPA (0.5 μM) leads to inhibition of spike generation in a spontaneously firing N1E-115 cell. Perforated patch-clamp recording under current-clamp conditions. Spontaneous firing is seen to resume after full recovery of the depolarization. Depolarization was accompanied by rapid growth cone collapse and neurite retraction Current Biology 2001 11, 121-124DOI: (10.1016/S0960-9822(01)00030-6)