Stephen J. Tucker

The essential role of the Walker A motifs of SUR1 in K‐ATP channel activation by Mg‐ADP and diazoxide

The ATP‐sensitive K‐channel (K‐ATP channel) plays a key role in insulin secretion from pancreatic β‐cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg‐ADP, which inhibit and potentiate channel activity, respectively. The β‐cell K‐ATP channel consists of a pore‐forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (WA) motifs of the first (K719A) and second (K1384M) nucleotide‐binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the WA lysine of NBD1 (but not NBD2) is essential for activation of K‐ATP currents by diazoxide. The potentiatory effects of Mg‐ADP required the presence of the WA lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild‐type currents. Metabolic inhibition led to activation of wild‐type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K‐ATP channel activity.

Molecular determinants of KATP channel inhibition by ATP

ATP‐sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore‐forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg‐nucleotides complicates analysis of nucleotide inhibition of Kir6.2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP‐sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the β‐phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N‐terminus preceding, and the C‐terminus immediately following, the transmembrane domains. Some mutations in the C‐terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single‐channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C‐terminus in KATP channel gating.

A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura

Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms. A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia. We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migraine-salient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele. These results therefore support a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target.

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co‐injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co‐expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4‐5 or 4‐5‐4‐5, but not when they were connected 4‐4‐5‐5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4‐5‐4‐5. Further, the relative order of subunits within the channel contributes to their functional properties.

The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation.

1 We have examined the mechanism by which nucleotides modulate the tolbutamide block of the β‐cell ATP‐sensitive K+ channel (KATP channel), using wild‐type and mutant KATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore‐forming (Kir6.2) subunits. 2 The dose–response relation for tolbutamide block of wild‐type KATP currents in the absence of nucleotide showed both a high‐affinity (Ki=2.0 μm) and a low‐affinity (Ki=1.8 mm) site. 3 The dose–response relation for tolbutamide block of Kir6.2ΔC36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low‐affinity site (Ki=1.7 mm). This indicates that the high‐affinity site resides on SUR1, whereas the low‐affinity site is located on Kir6.2. 4 ADP (100 μM) had a dual effect on wild‐type KATP currents: the nucleotide enhanced the current in the presence of Mg2+, but was inhibitory in the absence of Mg2+. Kir6.2ΔC36 currents were blocked by 100 μM ADP in the presence of Mg2+. 5 For wild‐type KATP currents, the blocking effect of 0.5 mm tolbutamide appeared greater in the presence of 100 μm MgADP (84 ± 2%) than in its absence (59 ± 4%). When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6 The ki for tolbutamide interaction with either the high‐ or low‐affinity site was unaffected by 100 μM MgADP, for both wild‐type and K719A‐K1384M currents. 7 MgGDP (100μM) enhanced wild‐type KATP currents and was without effect on K719A– K1384M currents. It did not affect the ki for tolbutamide block at either the high‐ or low‐affinity site. 8 Our results indicate that interaction of tolbutamide with the high‐affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the KATP channel.

K2P Channel Gating Mechanisms Revealed by Structures of Trek-2 and a Complex with Prozac

A sensitive regulator of cellular potassium A class of potassium channels called K2P channels modulates resting membrane potential in most cells. The channels are regulated by multiple ligands, including the antidepressant drug Prozac, as well as factors such as mechanical stretch and voltage. Dong et al. determined the structure of the human K2P channel, TREK-2, in two conformations and bound to a metabolite of Prozac. The structures show how ligand binding or mechanical stretch might induce switching between the states. Although both states have open channels, one appears primed for gating. A Prozac metabolite binds to the primed state and prevents conformational switching. K2P channels are not a target of Prozac, but their inhibition may contribute to side effects. Science, this issue p. 1256 Crystal structures clarify how a two-pore potassium channel is regulated by diverse stimuli. TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Differential pH sensitivity of Kir4.1 and Kir4.2 potassium channels and their modulation by heteropolymerisation with Kir5.1

1 The inwardly rectifying potassium channel Kir5.1 appears to form functional channels only by coexpression with either Kir4.1 or Kir4.2. Kir4.1‐Kir5.1 heteromeric channels have been shown to exist in vivo in renal tubular epithelia. However, Kir5.1 is expressed in many other tissues where Kir4.1 is not found. Using Kir5.1‐specific antibodies we have localised Kir5.1 expression in the pancreas, a tissue where Kir4.2 is also highly expressed. 2 Heteromeric Kir5.1‐Kir4.1 channels are significantly more sensitive to intracellular acidification than Kir4.1 currents. We demonstrate that this increased sensitivity is primarily due to modulation of the intrinsic Kir4.1 pH sensitivity by Kir5.1. 3 Kir4.2 was found to be significantly more pH sensitive (pKa= 7.1) than Kir4.1 (pKa= 5.99) due to an additional pH‐sensing mechanism involving the C‐terminus. As a result, coexpression with Kir5.1 does not cause a major shift in the pH sensitivity of the heteromeric Kir4.2‐Kir5.1 channel. 4 Cell‐attached single channel analysis of Kir4.2 revealed a channel with a high open probability (Po > 0.9) and single channel conductance of ˜25 pS, whilst coexpression with Kir5.1 produced novel bursting channels (Po < 0.3) and a principal conductance of ˜54 pS with several subconductance states. 5 These results indicate that Kir5.1 may form heteromeric channels with Kir4.2 in tissues where Kir4.1 is not expressed (e.g. pancreas) and that these novel channels are likely to be regulated by changes in intracellular pH. In addition, the extreme pH sensitivity of Kir4.2 has implications for the role of this subunit as a homotetrameric channel.

Inward rectification in KATP channels: a pH switch in the pore.

Inward‐rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage‐dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage‐dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine‐mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward‐rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH–dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C–terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH‐dependent rectification. This pH‐dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH.

Involvement of the N‐terminus of Kir6.2 in coupling to the sulphonylurea receptor

1 ATP‐sensitive potassium (KATP) channels are composed of pore‐forming Kir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high‐affinity site on SUR1 and a low‐affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2 We examined the effect of N‐terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild‐type or mutant Kir6.2/SUR1. 3 A 14 amino acid N‐terminal deletion (ΔN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2ΔC26, expressed in the absence of SUR1. Thus, the N‐terminal deletion does not alter the intrinsic properties of Kir6.2. 4 When Kir6.2ΔN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po= 0·7) and a lower ATP sensitivity (Ki= 196 μM) than wild‐type (Kir6.2/SUR1) channels (Po= 0·32, Ki= 28 μM). High‐affinity tolbutamide block was also abolished. 5 Truncation of five or nine amino acids from the N‐terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high‐affinity tolbutamide block, although to a lesser extent than for the ΔN14 deletion. Site‐directed mutagenesis suggests that hydrophobic residues in Kir6.2 may be involved in this effect. 6 The reduced ATP sensitivity of Kir6.2ΔN14 may be explained by the increased Po. However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6.2ΔN14/SUR1‐K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2‐C166S/SUR1‐K719A,K1385M currents (which in the absence of ATP have a Po of > 0·8 and are not blocked by tolbutamide). Thus the N‐terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore.

Involvement of the N‐terminus of Kir6.2 in the inhibition of the KATP channel by ATP

1 ATP‐sensitive potassium (KATP) channels are composed of pore‐forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2ΔC26, expresses ATP‐sensitive channels in the absence of SUR1, suggesting the ATP‐inhibitory site lies on the Kir6.2 subunit. 2 We examined the effect on the channel ATP sensitivity of mutating the arginine residue at position 50 (R50) in the N‐terminus of Kir6.2, by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild‐type or mutant Kir6.2ΔC26. 3 Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 μm to 800 μm, 1.1 mm and 3.8 mm, respectively. The single‐channel conductance and kinetics were unaffected by any of these mutations. Mutation to glutamate, lysine, asparagine, glutamine or leucine had a smaller effect (Ki, ∼300–400 μm). The results indicate that the side chain of the arginine residue at position 50 is unlikely to contribute directly to the binding site for ATP, and suggest it may affect ATP inhibition by allosteric interactions. 4 Mutation of the isoleucine residue at position 49 to glycine (I49G) reduced the channel ATP sensitivity, while the mutation of the glutamate residue at position 51 to glycine (E51G) did not. 5 When a mutation in the N‐terminus of Kir6.2ΔC26 that alters ATP sensitivity (R50S; Ki, 800 μm) was combined with one in the C‐terminus (E179Q; Ki, 300 μm), the Ki for the apparent ATP sensitivity was increased to 2.8 mm. The Hill coefficient was also increased. This suggests that the N‐ and C‐termini of Kir6.2 may co‐operate to influence channel closure by ATP.