Christina Schwanstecher

Potassium channel openers require ATP to bind to and act through sulfonylurea receptors

KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP‐binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic β‐cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular‐smooth‐muscle‐type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non‐hydrolysable ATP‐analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide‐binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C‐terminus of SUR affects KCO affinity with SUR2B ∼ SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5–8‐fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.

IDENTIFICATION OF THE POTASSIUM CHANNEL OPENER SITE ON SULFONYLUREA RECEPTORS

Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of KATPchannels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic β-cell receptor, SUR1, imparts high sensitivity to hypoglycemic sulfonylureas (SUs;e.g. glibenclamide) and low to potassium channel openers (KCOs; e.g. P1075), whereas the opposite drug sensitivities are conferred by cardiovascular receptors, SUR2A and SUR2B. By exchanging domains between SUR1 and SUR2B, we identify two regions (KCO I: Thr1059–Leu1087 and KCO II: Arg1218–Asn1320; rat SUR2 numbering) within the second set of transmembrane domains (TMDII) as critical for KCO binding. Swapping both regions reconstitutes KCO affinities and sensitivities of the donor SUR isoform. High glibenclamide affinity of SUR1 is not reduced by transfer of KCO I plus II from SUR2B, demonstrating that high SU and KCO affinity can coexist in the same SUR molecule. Consistently, high SU affinity was imparted on SUR2B by substituting the region separating KCO I and II (Ile1088–Val1217) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr1059–Asn1320) in TMDII of SURs.

Tolbutamide- and diazoxide-sensitive K+ channel in neurons of substantia nigra pars reticulata

SummarySingle-channel K+ currents were recorded in cell-attached patches from slices of rat substantia nigra. On the somata of neurons in the caudal half of the substantia nigra pars reticulata a K+ selective channel with a unitary conductance of 71 pS (154 mmol/l K+ in pipette filling solution) was identified. The channel was activated both by application of diazoxide (300 μmol/l) and by energy-depleting conditions (200 μmol/l cyanide) and was reversibly blocked by tolbutamide (0.1–1 mmol/l). It is concluded that neurons in the substantia nigra pars reticulata of the rat contain a typical ATP-sensitive K+ channel the activity of which can be modulated by diazoxide and sulfonylureas.

Location of the sulphonylurea receptor at the cytoplasmic face of the β‐cell membrane

1 In insulin‐secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH7.4 compound IV is only present in charged form. 2 Lipid solubility declined in the order tolbutamide > compound III > compound IV. 3 The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT‐cells (pancreatic β‐cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III. 4 In mouse pancreatic β‐cells, drug concentrations inhibiting adenosine 5′‐triphosphate‐sensitive K+ channels (KATP‐channels) half‐maximally (EC50) were determined by use of the patch‐clamp technique. When the drugs were applied to the extracellular side of outside‐out or the intracellular side of inside‐out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide. 5 In mouse pancreatic β‐cells, measurement of KATP‐channel activity in cell‐attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside‐out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly. 6 It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the β‐cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.

Diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic β-cells

1 . In mouse pancreatic β‐cells the regulation of the diazoxide‐sensitivity of the adenosine 5′‐triphosphate‐dependent K+ channel (K‐ATP‐channel) was examined by use of the patch‐clamp technique. 2 . In intact β‐cells incubated at 37°C in the presence of 3 mm d‐glucose, diazoxide did not affect the single channel conductance but stimulated channel‐opening activity. Diazoxide produced half‐maximal effects at 82 μm and 13 fold activation at maximally effective concentrations (300–400 μm). The response to diazoxide (300 μm) was not completely suppressed by saturating tolbutamide concentrations (1 or 5 mm). 3 . Inside‐out patch‐clamp experiments were carried out using an experimental protocol favouring phosphorylation of membrane proteins. Under these conditions diazoxide was ineffective in the absence of any nucleotides, weakly effective in the presence of MgATP (26 or 87 μm) and strongly effective in the presence of the Mg complexes of adenosine 5′‐diphosphate, 2′‐deoxyadenosine 5′‐diphosphate or guanosine 5′diphosphate (MgADP, MgdADP or MgGDP). 4 . In inside‐out patches exposed to nucleotide‐free solutions, saturating concentrations of tolbutamide did not cause complete block of K‐ATP‐channels. When the channels were activated by MgdADP (48 μm), tolbutamide was even less effective. Sensitization of MgdADP‐induced channel activation by diazoxide further weakened the effects of tolbutamide. 5 . Diazoxide (50 or 300 μm) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2+ and ADP (1 mm). 6. In the presence of Mg2+, the K‐ATP‐channel‐blocking potency of cytosolic ATP decreased in the order inside‐out > outside‐out > whole‐cell configuration of the patch‐clamp technique. 7. It is concluded that the K‐ATP‐channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.

Interaction of tolbutamide and cytosolic nucleotides in controlling the ATP-sensitive K+ channel in mouse β-cells

1 In mouse pancreatic β‐cells the role of cytosolic nucleotides in the regulation of the sulphonylurea sensitivity of the adenosine 5′‐triphosphate‐sensitive K+ channel (KATP‐channel) was examined. Patch‐clamp experiments with excised inside‐out membrane patches were carried out using an experimental protocol favouring phosphorylation of membrane proteins. 2 In the absence of Mg2+, the KATP‐channel‐inhibiting potency of cytosolic nucleotides decreased in the order ATP = adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS) > adenosine 5′‐diphosphate (ADP) > adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS) = adenylyl‐imidodiphosphate (AMP‐PNP) > 2′‐deoxyadenosine 5′‐triphosphate (dATP) > uridine 5′‐triphosphate (UTP) > 2′‐deoxyadenosine 5′‐diphosphate (dADP) > guanosine 5′‐triphosphate (GTP) > guanosine 5′‐diphosphate (GDP) > uridine 5′‐diphosphate (UDP). 3 In the presence of Mg2+, the inhibitory potency of cytosolic nucleotides decreased in the order ATPγS > ATP > AMP‐PNP > ADPβS > dATP > UTP. In the presence of Mg2+, the KATP‐channels were activated by dADP, GTP, GDP and UDP. 4 Tolbutamide inhibited the KATP‐channels not only in the presence but also in the prolonged absence of Mg2+. In nucleotide‐free solutions, the potency of tolbutamide was very low. When about half of the KATP‐channel activity was inhibited by ATP, AMP‐PNP, ADPβS or ADP (absence of Mg2+), the potency of tolbutamide was increased. 5 Tolbutamide (100 μm) slightly enhanced the channel‐inhibiting potency of AMP‐PNP and inhibited the channel‐activating effect of MgGDP in a non‐competitive manner. 6 Channel activation by MgGDP (0.5 mM) competitively antagonized the inhibitory responses to AMP‐PNP (1 μm‐1 mM). This effect of GDP was neutralized by tolbutamide (100 μm). 7 The stimulatory effect of 0.5 mM MgGDP was neutralized by 200 μm AMP‐PNP. Under these conditions the potency of tolbutamide was much higher than in the presence of 0.5 mM MgGDP alone or in the absence of any nucleotides. 8 dADP (0.3–1 mM) increased the potency of tolbutamide. Additional application of 200 μm AMP‐PNP caused a further increase in the potency of tolbutamide. 9 In conclusion, in the simultaneous presence of inhibitory and stimulatory nucleotides, binding of sulphonylureas to their receptor causes direct inhibition of channel activity, non‐competitive inhibition of the action of stimulatory nucleotides and interruption of the competitive interaction between stimulatory and inhibitory nucleotides. The latter effect increases the proportion of KATP‐channels staying in the nucleotide‐blocked state. In addition, this state potentiates the direct effect of sulphonylureas.

Identification of an ATP-sensitive K+ channel in spiny neurons of rat caudate nucleus

On the somata of GABAergic spiny neurons in the caudate nucleus of the rat an ATP-sensitive K+ channel (KATP-channel) was identified. The KATP-currents in cell-attached patches were activated both by energy-depleting conditions (200 μM cyanide) and by diazoxide (300 μM) and were reversibly blocked by tolbutamide (EC50=5 μM). In inside-out patch membranes both ATP (1 mM) and its non-hydrolyzable analog AMP-PNP (adenylylimidodiphosphate; EC50=27μM) reversibly inhibited channel activity. These results demonstrate that the KATP-channel in spiny neurons displays properties characteristic of the KATP-channel in hippocampal, neocortical and nigral neurons and in pancreatic ß-cells.