The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells

Figures

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  Figure 1. Strucutre of transmembrane KATP channels. Ribbon representation of superimposed Kir6.1 homology model (red) and Kir6.2 cryo-EM structure 6BAA (blue) complexed with SUR subunits, SUR2 homology model (orange), and SUR1 cryo-EM structure 6BAA (cyan).

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  Figure 2. Effect of norbormide on KATP currents of isolated rat tail artery myocytes. (A) Representative whole-cell recordings of inward currents elicited by 10 µM pinacidil at a V h of −50 mV. The effect of 5 µM norbormide as well as of 10 µM glibenclamide is shown. (B) Pinacidil (10 µM) activated glibenclamide-sensitive KATP currents, which were inhibited by either (C) 5 µM or (D) 50 µM norbormide. Columns are mean ± SEM (n = 5–7). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test for paired samples.

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  Figure 3. Effect of norbormide on KATP currents of isolated rat and mouse tail artery or rat fundus myocytes. (A and B) Pinacidil (10 µM) activated glibenclamide-sensitive rat tail artery myocytes KATP currents, which were inhibited by either (A) 50 µM exo-norbormide (NRB) or (B) 50 µM endo NRB. (C and D) Pinacidil (10 µM) activated glibenclamide-sensitive (C) mouse tail artery and (D) rat gastric fundus myocytes KATP currents, which were inhibited by 50 µM NRB. Columns are mean ± SEM (n = 4–5). *P < 0.05, **P < 0.01, Student’s t test for paired samples.

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  Figure 4. Effect of norbormide on KATP currents of INS-1 cell. KATP currents were recorded at a V h of −70 mV during 500-ms pulses to −60 mV (white columns) and −80 mV (black columns) in the absence (control) or presence of 50 µM norbormide (NRB) as well as NRB plus 10 µM glibenclamide (glib). Columns are mean ± SEM (n = 7). Insets: average traces of original KATP currents, recorded from seven cells, during 500-ms pulses to −60 mV (left) or −80 mV (glib) in the absence (control) or presence of NRB as well as NRB plus glibenclamide. P < 0.001, ANOVA and Dunnett post test.

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  Figure 5. Effects of norbormide and glibenclamide on the resting tone of rat caudal artery rings and gastric fundus strips. (A–D) Rat caudal artery rings. (A) Original recording showing the contractile effect of a maximal concentration of norbormide (50 µM). The addition of 10 µM pinacidil did not affect active muscle tone (compared to panel B). (B) Control experiment, performed in parallel with that represented in (A), showing that norbormide-induced contraction spontaneously relaxes after the plateau phase, thus suggesting that the relaxation observed in (A) is not pinacidil-dependent. (C) Original recording showing the contractile effect of a low concentration of norbormide (5 µM). The addition of 10 µM pinacidil caused a partial relaxation of the active muscle tone. Inset: effect of pinacidil on rat caudal rings precontracted with 5 µM norbormide. On the ordinate scale, contraction is reported in g (n = 6; *P < 0.05, Student’s t test for paired samples). (D) Glibenclamide, up to 200 µM, was unable to affect ring resting tone. (A–D) The contraction induced by 90 mM KCl and/or 10 µM phenylephrine is also shown. Neither (E) glibenclamide nor (F) norbormide contracted rat gastric fundus…

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  Figure 6. Glibenclamide and norbormide binding site. (A and B) Inhibited SUR2 cartoon structure (PDB accession number 6BAA) showing only the interactions between residues’ binding site (cyan sticks) and (A) glibenclamide (purple sticks) or (B) norbormide (blue sticks). (C and D) SUR1 cartoon model structure, showing only the interactions between residues’ binding site (orange sticks) and (C) glibenclamide (purple sticks) or (D) norbormide (blue sticks).

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  Figure 7. Sequence alignment results between R. norvegicus SUR2 and the template sequence of R. norvegicus SUR1. Identical and positively conserved amino acids (marked with a star and colon, respectively) are highlighted in colors according to the property of the corresponding query residue in the alignment. Green arrows highlight all the amino acids involved in glibenclamide and norbormide binding regions. Red arrows: non-conserved amino acids engaged in interaction network.