Kinsella JM , Laidlaw HA , Tang T , Harvey J , Sutherland C , Ashford ML .

068692 Wellcome Trust , WT068692 Wellcome Trust , Wellcome Trust

	Figure 1. Structure of BVT.12777 ([2-(hydrazinoiminomethyl)-hydrazino] acetic acid)
	Figure 2. BVT.12777 activates a tolbutamide-sensitive K+ current A, the upper trace shows a current clamp recording of a CRI-G1 cell following dialysis with a 5 mM ATP-containing solution. In this and subsequent current clamp figures the trace begins approximately 5 min after formation of the whole-cell configuration. Application of BVT.12777 (100 μM) for the time indicated hyperpolarized the cell from -50 mV to -76 mV, an action readily reversed by tolbutamide (100 μM), which returned membrane potential to -54 mV. Washout of all drugs from the bath resulted in a membrane potential of -70 mV, indicating the lack of reversibility of BVT.12777. The lower plot is the current-voltage relationship for the voltage clamped currents. Cells were voltage clamped at -50 mV and 10 mV steps of 100 ms duration were applied every 200 ms (range -120 to -30 mV). BVT.12777 increased the membrane conductance relative to control and tolbutamide reversed this BVT.12777-induced conductance increase with a reversal potential of -78 mV. B, cell-attached recording from a CRI-G1 cell, at 10 mV applied to the recording pipette. Single channel openings are shown as downward deflections. Addition of 100 μM BVT.12777 induced an increase in channel activity (Nf.Po) from 0.17 in control to 0.31, and 1.25 at 10 and 20 minutes respectively, after BVT addition. Application of 100 μM tolbutamide induced a substantial inhibition of activity (to 0.02), which was reversed on washout of all drugs, with activity increasing to 0.74. The symbol C refers to the closed state of the channel in this and subsequent figures. C, diary plot of Nf.Po against time from cell-attached experiments in the presence and absence of BVT.12777, where channel activity was calculated every 2 minutes. Each point is the mean of 4–7 separate determinations.
	Figure 3. BVT.12777 activates KATP channels in inside-out patches Continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited channel activity by >90%, demonstrating KATP identity. Addition of 100 μM BVT.12777, in the presence of 100 μM MgATP to the cytoplasmic aspect of the patch resulted in KATP channel activation. Nf.Po values were 2.96 (control, after first MgATP challenge), and 0.25 in the presence of MgATP, which increased to 0.72, 1.06 and 2.74 at 5, 10 and 20 minutes respectively, after BVT.12777 addition.
	Figure 4. Wortmannin does not inhibit BVT.12777 activation of KATP A, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Application of BVT.12777 (200 μM), in the continued presence of wortmannin hyperpolarized the cell from -46 to -77 mV. Tolbutamide (100 μM), applied after the BVT-induced hyperpolarization, recovered the membrane potential (to -40 mV). B, cell-attached recordings from CRI-G1 cells, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Upper trace; in the continued presence of wortmannin, Nf.Po was 0.01 and 0.03 after 5 and 30 minutes respectively. Lower trace, application of BVT.12777 (100 μM) to cell-attached recording in the presence of 10 nM wortmannin resulted in KATP activation, with Nf.Po values of 0.01, 0.12 and 0.27 prior to, and 10 and 30 minutes after, BVT.12777, respectively. Addition of 100 nM wortmannin did not inhibit channel activity.
	Figure 5. Phalloidin prevents BVT.12777 activation of KATP A, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of BVT.12777 (200 μM) had no effect on the membrane potential of the cell (-41 mV) in the presence of phalloidin. B, continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited Nf.Po from 1.25 to 0.02. Addition of 10 μM phalloidin and subsequently 100 μM BVT.12777, in the presence of 100 μM MgATP, to the cytoplasmic aspect of the patch resulted in no effect on KATP, with Nf.Po values of 0.01 and 0.03 respectively. C, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of diazoxide (200 μM) induced rapid cell membrane hyperpolarization, from -55 to -72 mV, an action reversed (to -45 mV) by tolbutamide (100 μM).
	Figure 6. BVT.12777 disrupts the actin cytoskeleton A, images of rhodamine-conjugated phalloidin fluorescence in CRI-G1 cells in control conditions and following incubation with leptin (10 nM), BVT.12777 (100 μM) or diazoxide (200 μM) for 30 minutes. All panels show representative X-Y images. Note the marked reduction in phalloidin staining in cells pre-treated with leptin or BVT.12777, and not diazoxide. Scale bars are 50 μm. B, histogram comparing the normalised fluorescence intensity relative to control in the membrane periphery of randomly selected CRI-G1 cells for each condition; (control (n = 13; cells = 195), 10 nM leptin (n = 6; cells = 90), BVT.12777 (n = 6; cells = 90) and 200 μM diazoxide (n = 4, cells = 60). Error bars indicate s.e.m. and * significance of P < 0.001.

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