Regulation of an ATP-conductive large-conductance anion channel and swelling-induced ATP release by arachidonic acid

Abstract

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume-dependent ATP-conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling-induced ATP release in the physiological concentration range with K(d) of 4- 6 microM. The former effect observed in the whole-cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate-metabolizing oxygenases, and by the observation that they were mimicked by cis-unsaturated fatty acids, which are not substrates for oxygenases. A membrane-impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non-charged arachidonate analogues as well as trans-unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonate's negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (K(d) of 4- 5 microM) and reduced unitary conductance (K(d) of 13-14 microM) without affecting voltage dependence of open probability. ATP(4-)-conducting inward currents measured in the presence of 100 mM ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling-induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.

Figure 1. Swelling-activated, phloretin-insensitive chloride currents were inhibited by arachidonic acid in C127 cells

A, representative record during application of alternating pulses from 0 to ±25 mV (every 10 s) or of step pulses from −50 to +50 in 10 mV increments before (a) and after (b) application of arachidonic acid. Horizontal bars indicate the time of application of hypotonic solution containing 300 μm phloretin in the absence and presence of arachidonic acid (20 μm). B, expanded traces of current responses to step pulses recorded at the time indicated by a and b in A. The step-pulse protocol is shown in the top panel. C, current-voltage relationships measured at the beginning of the pulses in the absence (control; ○) and presence of 20 μm arachidonic acid (•). Points plotted are mean ± s.e.m. The whole-cell currents were normalized by the cell capacitance (n = 4). The remaining current in the presence of arachidonic acid had a linear I-V relationship and a reversal potential of around 3 mV, suggesting a non-selective leak current.

Figure 2. Macro-patch currents activated in inside-out (A) and outside-out patches (B) were reversibly suppressed by arachidonic acid in a dose-dependent manner

a, representative mean macropatch currents during application of alternating pulses from 0 to ±25 mV (every 10 s) are presented. Horizontal bars indicate the time of application of arachidonic acid (20 μm). b, concentration dependence of arachidonate effects on VDACL currents recorded at ±25 mV, plotted as mean ± s.e.m. All currents measured in the presence of arachidonate were normalized to the respective currents measured before arachidonate application (I_o). The inside-out data were fitted to eqn (1) with K_d / 3.9 ± 0.21 μm and 4.44 ± 0.23 μm for outward and inward currents, respectively (n = 5-11). The outside-out data were fitted to eqn (1) with K_d = 4.99 ± 0.44 μm and 4.54 ± 0.37 μm for outward and inward currents, respectively (n = 5-7). The Hill coefficient was 2.

Figure 3. Effects of a membrane-impermeant analogue of arachidonic acid, arachidonyl coenzyme A (20 μm), on VDACL currents recorded at ±25 mV from inside-out (A) and outside-out patches (B)

Representative mean macropatch currents during application of alternating pulses from 0 to ±25 mV (every 10 s) are presented. Horizontal bars indicate the time of application of arachidonyl coenzyme A (20 μm). C, macropatch currents in inside-out and outside-out patches in the presence of arachidonyl coenzyme A (20 μm), normalized to those measured before application of arachidonyl coenzyme A (I_o). * Significantly different from control at P < 0.001.

Figure 4. Suppression of macropatch currents in the inside-out mode by arachidonic acid was not affected by inhibitors of oxygenases that are involved in arachidonic acid metabolism

A, effect of arachidonic acid in the presence of 20 μm indomethacin. B, effect of arachidonic acid in the presence of 20 μm NDGA. C, effect of arachidonic acid in the presence of 20 μm clotrimazole. D, inhibitory effect of arachidonic acid in the absence or presence of oxygenase inhibitors (mean ± s.e.m.). Percentage inhibition of macropatch currents by arachidonic acid in the presence or absence of oxygenase inhibitors was calculated from currents measured before application of arachidonic acid.

Figure 5. Role of the hydrophobic tail and polar head in inhibition of VDACL currents by arachidonic acid

A, relative effects of cis-unsaturated (arachidonic, oleic and linoleic), trans-unsaturated (elaidic) and saturated (palmitic) fatty acids (all 20 μm) on macropatch currents recorded in the inside-out mode at ±25 mV. * Significantly different from control at P < 0.001. B, concentration-dependent inhibition of macropatch currents by oleic acid (squares) and linoleic acid (triangles). Open symbols are data for +25 mV and filled symbols are for −25 mV (mean ± s.e.m.). The data were fitted to eqn (1) with K_d / 4.02 ± 0.21 μm for +25 mV and K_d = 4.19 ± 0.34 μm for −25 mV in the presence of oleic acid, and K_d = 9.3 ± 1.2 μm for +25 mV and K_d = 10.0 ± 1.3 μm for −25 mV in the presence of linoleic acid (n = 5-7). The Hill coefficient was 2. C, effects of non-charged arachidonic acid analogues, arachidonyl alcohol and methyl ester, and a non-charged oleate analogue, oleyl alcohol, on VDACL macropatch currents in inside-out patches. Data were normalized to the mean current measured before application of drugs (I_o).

Figure 6. Effect of arachidonic acid added to the intracellular side on single VDACL channel currents recorded from inside-out patches

A, representative current traces recorded during application of a step pulse from 0 to ±25 mV in the absence (control) and presence of 10 μm arachidonic acid. The pulse protocol is shown at the top of the traces. B, amplitude histograms of current traces presented in A. C, unitary current-voltage relationships in the absence (control; ○) and presence of 10 μm arachidonic acid (•). Mean single-channel conductance was 405.6 ± 6.4 pS for control and 249.2 ± 7.2 pS with 10 μm arachidonic acid (P < 0.05, n = 5-13). D, concentration-response curves of arachidonic acid effects on VDACL single-channel amplitude measured at ±25 mV. The data were fitted to eqn (1) with K_d = 13.8 ± 1.6 μm and 14.0 ± 1.1 μm for outward and inward currents, respectively (n = 5-9). The Hill coefficient was 2. Points plotted are mean ± s.e.m.

Figure 7. Fast reducing effect of arachidonic acid on single-channel current amplitude

A, representative current traces recorded from an inside-out patch during application of a test pulse from 0 to ±25 mV (protocol is shown at the top of the traces) by sampling at 10 kHz and filtering at 5 kHz in the absence (control; upper record) and presence of 10 μm arachidonic acid (lower record). B, amplitude histograms for data presented in A.

Figure 8. Effect of arachidonic acid on mean macropatch currents, single-channel amplitude and open-channel probability

Fractional macropatch and single-channel currents in inside-out patches in the presence of 10 μm arachidonic acid without (A) and with (B) a cocktail of three oxygenase inhibitors (indomethacin, NDGA and clotrimazole, each 20 μm). Data were normalized to the mean current measured before application of arachidonic acid (I_o) and plotted as mean ± s.e.m.C, voltage dependence of steady-state open-channel probability. The data represent the ensemble-averaged current of 11 consecutive current responses to ramp pulses (from −50 mV to +50 mV at 10 mV s⁻¹ rate) in the absence (control; continuous line) and presence of 5 μm arachidonic acid (○). The patch contained 5–6 channels. The P_o values were calculated as P_o / (I/V)/G_max, where I is the patch current, V is voltage and G_max is the maximal patch conductance at 0 mV.

Figure 9. Arachidonic acid sensitivity of ATP current through VDACL channels

A, mean patch currents in an inside-out patch measured at ±50 mV. All anions in the bath were replaced with 100 mm ATP. Upper horizontal bar represents the time of arachidonic acid (20 μm) application. Data represent 12 similar experiments. a, b and c indicate the times when the traces in B were recorded. B, current traces recorded during application of alternating pulses from 0 to ±50 mV (protocol is shown at the top of the traces) before application of arachidonic acid (a), in the presence of 20 μm arachidonic acid (b), and after washout (c). C, representative inward ATP^4- currents recorded at −50 mV from three different patches before (left: control) and during (right) application of 20 μm arachidonic acid.

Figure 10. Effects of arachidonic acid and other fatty acids on swelling-induced ATP release from C127 cells

A, swelling-induced ATP release as a function of medium tonicity in the absence (control; ○) and presence of 20 μm arachidonic acid (•). B, ATP release at 127.5 mosmol (kg H₂O)⁻¹ as a function of arachidonic acid concentration in the absence (control: open circles) and presence of inhibitors of arachidonic acid metabolism (indomethacin, NDGA, clotrimazole, 20 μm each; •). The data were fitted to eqn (2) with K_d / 6.2 ± 0.3 μm for control and K_d = 4.4 ± 1.2 μm for ATP release in the presence of metabolic inhibitors (n = 6). The Hill coefficient was 2. C, relative effects of cis-unsaturated (arachidonic, oleic and linoleic), trans-unsaturated (elaidic) and saturated (palmitic) fatty acids (all 20 μm) on ATP release measured at 127.5 mosmol (kg H₂O)⁻¹. n = 6. Points plotted are mean ± s.e.m. * Significantly different from control at P < 0.01.