Ramu Y et al. (2018), A novel high-affinity inhibitor against the hum...

XB-ART-56318

J Gen Physiol 2018 Jul 02;1507:969-976. doi: 10.1085/jgp.201812017.

Show Gene links Show Anatomy links

A novel high-affinity inhibitor against the human ATP-sensitive Kir6.2 channel.

Ramu Y , Xu Y , Lu Z .

???displayArticle.abstract???
The adenosine triphosphate (ATP)-sensitive (KATP) channels in pancreatic β cells couple the blood glucose level to insulin secretion. KATP channels in pancreatic β cells comprise the pore-forming Kir6.2 and the modulatory sulfonylurea receptor 1 (SUR1) subunits. Currently, there is no high-affinity and relatively specific inhibitor for the Kir6.2 pore. The importance of developing such inhibitors is twofold. First, in many cases, the lack of such an inhibitor precludes an unambiguous determination of the Kir6.2's role in certain physiological and pathological processes. This problem is exacerbated because Kir6.2 knockout mice do not yield the expected phenotypes of hyperinsulinemia and hypoglycemia, which in part, may reflect developmental adaptation. Second, mutations in Kir6.2 or SUR1 that increase the KATP current cause permanent neonatal diabetes mellitus (PNDM). Many patients who have PNDM have been successfully treated with sulphonylureas, a common class of antidiabetic drugs that bind to SUR1 and indirectly inhibit Kir6.2, thereby promoting insulin secretion. However, some PNDM-causing mutations render KATP channels insensitive to sulphonylureas. Conceptually, because these mutations are located intracellularly, an inhibitor blocking the Kir6.2 pore from the extracellular side might provide another approach to this problem. Here, by screening the venoms from >200 animals against human Kir6.2 coexpressed with SUR1, we discovered a small protein of 54 residues (SpTx-1) that inhibits the KATP channel from the extracellular side. It inhibits the channel with a dissociation constant value of 15 nM in a relatively specific manner and with an apparent one-to-one stoichiometry. SpTx-1 evidently inhibits the channel by primarily targeting Kir6.2 rather than SUR1; it inhibits not only wild-type Kir6.2 coexpressed with SUR1 but also a Kir6.2 mutant expressed without SUR1. Importantly, SpTx-1 suppresses both sulfonylurea-sensitive and -insensitive, PNDM-causing Kir6.2 mutants. Thus, it will be a valuable tool to investigate the channel's physiological and biophysical properties and to test a new strategy for treating sulfonylurea-resistant PNDM.

???displayArticle.pubmedLink??? 29844136
???displayArticle.pmcLink??? PMC6028498
???displayArticle.link??? J Gen Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: abcc8 ins
GO keywords: sulfonylurea receptor activity

???attribute.lit??? ???displayArticles.show???

	Figure 1. Relative insensitivity of Kir6.2 to numerous known inhibitors for various types of K+ channels. Currents of Kir6.2 coexpressed with SUR1, recorded in 100 mM KCl-containing bath solution without (black traces) or with (red traces) the presence of 24 known K+ channel inhibitors (1 µM): agitoxin-1 (AgTx-1), agitoxin-2 (AgTx-2), apamin, blood depressing substance I (BDS-I), blood depressing substance II (BDS-II), chlorotoxin (CTx), α-dendrotoxin (α-DTx), β-dendrotoxin (β-DTX), gamma-dendrotoxin (γ-DTX), delta-dendrotoxin (δ-DTX), ergotoxin, heteropodatoxin (HpTx), iberiotoxin (IbTx), kaliotoxin (KTx), Leiurus quinquestriatus toxin 2 (Lq-2), margatoxin (MgTx), maurotoxin (MTx), mast cell degranulating peptide (MCDP), noxiustoxin (NTx), paxilline, scyllatoxin, slotoxin, stichodactyla toxin (ShK), and tertiapin-Q (TPNQ). In most cases, the pair of compared traces are superimposed. The currents were activated with 3 mM azide (Gribble et al., 1997) and elicited by stepping voltages from the holding potential 0 mV to −80 and then 80 mV before returning to 0 mV. The dotted lines indicate zero-current levels. In a few cases, a very small current occurred at the 0-mV holding potential, which indicates the fact that not all oocytes had an overall reversal potential about ∼0 mV in the presence of 100 mM extracellular K+.
	Figure 2. Purification of an inhibitor against Kir6.2. (A and B) Currents of Kir6.2 coexpressed with SUR1, recorded in the absence (black trace) or the presence (red trace) of 1:2,000 diluted venom (A) or ∼30 nM purified inhibitor from the venom (B). The dotted line indicates the zero-current level. (C) HPLC chromatograph (25-cm-long C18 column) of crude venom, where the sample was eluted with a linear methanol gradient of 1% per min at a 1-ml/min flow rate. The active peak is indicated by the arrow. (D) HPLC chromatograph (15-cm-long C18 column) of the active fraction collected from C, where sample was eluted with a methanol gradient of 0.2% per min at a 0.2-ml/min flow rate.
	Figure 3. Amino acid sequence of SpTx-1. The partial sequence of the purified inhibitor obtained from Edman degradation (A; each letter X indicates an ambiguous residue type that was subsequently determined to be the corresponding one shown in B), the full sequence deduced from cDNA sequencing (B), and the sequence with an extra N-terminal segment containing a 6-His-tag followed by a spacer sequence and then by the cleaving site of the protease TEV (C), where the His-tag and the TEV-cleaving sequence were colored maroon and cyan, respectively.
	Figure 4. Properties of synthetic and recombinant SpTx-1. (A and B) Currents of Kir6.2 coexpressed with SUR1, recorded in the absence (black trace) or presence (red trace) of 20 nM synthetic (A) and recombinant (B) SpTx-1. The dotted line indicates the zero-current level. (C) Fractions of currents plotted against the concentration of synthetic or recombinant SpTx-1. The curves superimposed on data correspond to the fits of an equation for a bimolecular reaction. The fitted Kd values are (1.53 ± 0.13) × 10−8 M (n = 6, synthetic) and (1.48 ± 0.12) × 10−8 M (n = 6, recombinant). (D) appkon (solid squares) and appkoff (solid circles) of current inhibition plotted against the concentration of recombinant SpTx-1 with the extra sequence shown in Fig 3 C. A linear fit yielded kon of (1.46 ± 0.42) × 107 M−1 s−1 (n = 5), and koff, calculated from the mean of appkoff at various SpTx-1 concentrations, was (1.23 ± 0.19) × 10−1 s−1 (n = 5). Data represent the mean ± SEM.
	Figure 5. Inhibition of the Kir6.2-ΔC26 mutant by SpTx-1. (A) Currents of the Kir6.2-ΔC26 mutant channels, recorded in the absence (black trace) or presence (red trace) of 10 nM synthetic SpTx-1. (B) Fractions of currents, recorded from WT and Kir6.2-ΔC26 mutant channels, are plotted against the concentration of recombinant SpTx-1. The curves superimposed on data correspond to the fits of an equation for a bimolecular reaction. The fitted Kd values are (1.53 ± 0.13) × 10−8 M (n = 6) for WT channels and (8.52 ± 0.37) × 10−9 M (n = 6) for the Kir6.2-ΔC26 mutant channels. Data represent the mean ± SEM.
	Figure 6. Selectivity of SpTx-1 among various types of Kir channels. (A–F) Currents of six types of Kir channels, recorded in the absence (black traces) or presence (red traces) of 250 nM synthetic SpTx-1.
	Figure 7. SpTx-1 sensitivity of Kir6.2 containing PNDM-causing mutations. (A) Currents of Kir6.2 with individual PNDM-causing mutations on the ΔC26 background without (black trace) or with (red trace) the presence of 10 nM recombinant SpTx-1. (B) Fractions of current, recorded from the Kir6.2-ΔC26 mutant channels with or without these PNDM-causing mutations, are plotted against the concentration of SpTx-1. The Kd values determined from the curve fitting are in nM: 8.5 ± 0.37 (Kir6.2-ΔC26 itself), 13.0 ± 3.5 (Q52R), 21 ± 2 (V59G), 11.6 ± 0.9 (R201C), 5.8 ± 0.8 (R201H), 11.3 ± 0.6 (I296L), and 11.6 ± 1.2 (G334D). n = 6–10. Data represent the mean ± SEM.

References [+] :

Aguilar-Bryan, Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. 1995, Pubmed