Figures and data in Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

Version of Record: February 27, 2020
Version of Record: February 6, 2020
Accepted Manuscript: January 7, 2020

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Samuel G Usher

Frances M Ashcroft

Michael C Puljung

(2020)
Nucleotide inhibition of the pancreatic ATP-sensitive K⁺ channel explored with patch-clamp fluorometry

eLife 9:e52775.

https://doi.org/10.7554/eLife.52775
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Figures
Tables
Additional files

4 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements

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A FRET assay to measure nucleotide binding to Kir6.2.

(A) Cartoon illustrating the topology of K_ATP. Two (of four) Kir6.2 and two (of four) SUR1 subunits are shown for clarity. The inhibitory nucleotide-binding site on Kir6.2 is shown in red; the …

Figure 1—figure supplement 1

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ANAP labelling is specific and only full-length Kir6.2 is expressed at the cell membrane.

(A) Confocal images of HEK-293T cells transfected with Kir6.2*-GFP + SUR1 (top panel) or Kir6.2-GFP + SUR1 (bottom panel). Cells were stained with Cell Mask Deep Red to label the plasma membrane. …

Figure 1—figure supplement 2

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Kir6.2*-GFP is functionally similar to Kir6.2-GFP.

A. Concentration-response curve for ATP inhibition of Kir6.2-GFP + SUR1 or Kir6.2*-GFP + SUR1, measured in excised, inside-out patches. The smooth curves are descriptive Hill fits to the data. …

Figure 2 with 3 supplements

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Simultaneous measurements of nucleotide binding and channel current.

(A) Brightfield and fluorescence images of a patch pipette and excised, inside out patch expressing Kir6.2*-GFP + SUR1, with the location of the centre of the spectrometer slit overlaid as a white, …

Figure 2—figure supplement 1

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Fixing $L$ does not affect estimates of $D$ and $K_{A}$ .

(A) Pairwise correlation plots of $L$ , $D$ and $K_{A}$ from the full MWC-type model fit to Kir6.2*-GFP + SUR1. (B) Pairwise correlation plots of $D$ and $K_{A}$ from the full MWC-type model with $L$ fixed to 0.8 …

Figure 2—figure supplement 2

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Model selection.

Fits to PCF data from Figure 2 with the full MWC-type model (A), single-binding model (B) and negative-cooperativity model (C) are shown on the left with the diagrammatic formulation of each model …

Figure 2—figure supplement 3

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Bleaching correction for PCF experiments.

(A) Raw ANAP fluorescence intensities (left) and corrected ANAP fluorescence intensities (right) from a representative PCF experiment with Kir6.2*-GFP + SUR1 plotted against the exposure time. The …

Figure 3 with 2 supplements

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Kir6.2-C166S disrupts current inhibition, not nucleotide binding.

(A) Cartoon (from PDB accession #6BAA) showing the location of Kir6.2-C166 (purple) relative to the inhibitory nucleotide binding site (TNP-ATP from PDB accession #5XW6 shown in red). W311 is shown …

Figure 3—figure supplement 1

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AN MWC-type model predicts a nucleotide-insensitive current plateau for Kir6.2-C166S.

A. Representative current trace from an excised patch expressing Kir6.2*,C166S-GFP + SUR1 exposed to triethylamine (shown as shades of red) and $10 mM$ ATP (shown in black). B. Concentration dependence …

Figure 3—figure supplement 2

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Fixing $L$ does not affect the other two parameters.

(A) Pairwise correlation plots of $L$ , $D$ and $K_{A}$ from the full MWC-type model fit to Kir6.2*-GFP + SUR1 and Kir6.2*,C166S-GFP + SUR1. (B) Pairwise correlation plots of $D$ and $K_{A}$ from the full …

Figure 4 with 4 supplements

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SUR1-K205 modulates both nucleotide affinity and inhibition of Kir6.2.

(A) Hydrophobic surface representation of Kir6.2 (yellow, PDB accession #6BAA) and SUR1 (blue, PDB accession #6PZI). Residue K205 on SUR1 is highlighted in pink. As this residue was built as an …

Figure 4—figure supplement 1

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SUR1 affects the apparent affinity for nucleotide binding to Kir6.2.

(A) Concentration dependence of TNP-ATP binding to unroofed membrane fragments expressing Kir6.2*-GFP without SUR1 (brown), expressed as quenching of ANAP fluorescence. The smooth curve is a …

Figure 4—figure supplement 2

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Fixing the $L$ parameter does not drastically affect the fits to the SUR1-K205A or SUR1-K205E data.

(A) Pairwise correlation plots of $L$ , $D$ and $K_{A}$ from the full MWC-type model fit to Kir6.2*-GFP co-expressed with wild-type SUR1, SUR1-K205A, and SUR1-K205E. (B) Pairwise correlation plots of $D$ and …

Figure 4—figure supplement 3

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Comparing the ability of each model to explain the data.

Fits for each construct with each model (MWC-type, single-binding, negative-cooperativity) are displayed with the solid curve representing the median fit, the shaded area representing the 95% …

Figure 4—figure supplement 4

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A neutral choice of priors allows for the best fits to the data.

(A) We generated more informative priors (dashed lines) based on our posterior probability distributions for Kir6.2*-GFP + SUR1 (in grey) by fitting the posterior distribution for each parameter …

Tables

Table 1

Hill fit parameters from unroofed membranes.

$E C_{50}$ values and their standard errors are reported as $\log_{10} M$ . $E C_{50}$ values are also provided as µM in parentheses.

Fluorescence quenching	Construct	Term	Estimate	Standard error
TNP-ATP	Kir6.2*-GFP+SUR1	$E C_{50}$	−4.59 (25.7)	0.05
	n = 18	$h$	0.82	0.05
		$E_{m a x}$	0.93	0.03
	Kir6.2*,G334D-GFP+SUR1	$E C_{50}$	−3.31 (490)	2.23
	n = 9	$h$	2.63	17.70
		$E_{m a x}$	0.08	0.26
	Kir6.2*,C166S-GFP+SUR1	$E C_{50}$	−4.50 (31.6)	0.05
	n = 12	$h$	0.92	0.08
		$E_{m a x}$	0.87	0.03
	Kir6.2*-GFP	$E C_{50}$	−4.42 (38.0)	0.05
	n = 14	$h$	0.83	0.05
		$E_{m a x}$	0.92	0.03

Table 2

Hill fit parameters from excised patches.

$E C_{50}$ and $I C_{50}$ values and their standard errors are reported as $\log_{10} M$ . $E C_{50}$ and $I C_{50}$ values are also provided as µM in parentheses.

Current inhibition	Construct	Term	Estimate	Standard error
ATP	Kir6.2-GFP + SUR1	$I C_{50}$	−4.20 (63.1)	0.07
	n = 3	$h$	1.28	0.21
		$I_{m a x}$	0.99	0.06
	Kir6.2-GFP	$I C_{50}$	−3.31 (490)	0.05
	n = 2	$h$	1.15	0.12
		$I_{m a x}$	0.93	0.03
	Kir6.2*-GFP + SUR1	$I C_{50}$	−4.10 (79.4)	0.06
	n = 4	$h$	1.42	0.21
		$I_{m a x}$	1.00	0.05
TNP-ATP	Kir6.2-GFP + SUR1	$I C_{50}$	−5.93 (1.17)	0.04
	n = 7	$h$	1.14	0.11
		$I_{m a x}$	0.97	0.02
	Kir6.2-GFP	$I C_{50}$	−3.56 (275)	0.64
	n = 3	h	1.09	0.85
		$I_{m a x}$	1.00	0.53
	Kir6.2*-GFP + SUR1	$I C_{50}$	−5.21 (6.17)	0.10
	n = 9	$h$	0.92	0.18
		$I_{m a x}$	0.96	0.05
	Kir6.2*,C166S-GFP + SUR1	$I C_{50}$	−3.11 (776)	0.23
	n = 6	$h$	1.35	1.16
		$I_{m a x}$	0.55	0.11
	Kir6.2*-GFP + SUR-K205A	$I C_{50}$	−3.78 (166)	0.45
	n = 9	$h$	0.75	0.30
		$I_{m a x}$	1.00	0.29
	Kir6.2*-GFP + SUR-K205E	$I C_{50}$	−3.20 (631)	2.15
	n = 9	$h$	0.79	0.84
		$I_{m a x}$	1.00	1.77
Fluorescence Quenching
TNP-ATP	Kir6.2*-GFP + SUR1	$E C_{50}$	−4.11 (77.6)	0.09
	n = 9	$h$	0.87	0.11
		$E_{m a x}$	1.00	0.06
	Kir6.2*,C166S-GFP + SUR1	$E C_{50}$	−4.17 (67.6)	0.23
	n = 6	$h$	0.84	0.27
		$E_{m a x}$	1.00	0.14
	Kir6.2*-GFP + SUR-K205A	$E C_{50}$	−3.69 (204)	0.42
	n = 9	$h$	0.73	0.25
		$E_{m a x}$	1.00	0.27
	Kir6.2*-GFP + SUR-K205E	$E C_{50}$	−3.37 (427)	1.10
	n = 9	$h$	0.74	0.47
		$E_{m a x}$	1.00	0.79

Table 3

Fitted parameters for the MWC-type models.

$L$ , $K_{A}$ and their associated quantiles are reported as $\log_{10}$ values.

Full MWC
Construct	Term	Estimate	2.5% Quantile	97.5% Quantile
Kir6.2*-GFP + SUR1	$L$	−1.05	−1.85	−0.45
n = 9	$D$	0.04	0.00	0.19
	$K_{A}$	4.32	4.21	4.44
Kir6.2*,C166S-GFP + SUR1	$L$	0.29	−1.04	1.41
n = 6	$D$	0.84	0.52	0.95
	$K_{A}$	4.18	3.93	4.47
Kir6.2*-GFP + SUR-K205A	$L$	−0.37	−1.34	0.41
n = 9	$D$	0.55	0.39	0.65
	$K_{A}$	3.76	3.59	3.95
Kir6.2*-GFP + SUR-K205E	$L$	−0.18	−1.25	0.70
n = 9	$D$	0.62	0.42	0.74
	$K_{A}$	3.40	3.21	3.62
Single-site
Kir6.2*-GFP + SUR1	$L$	−1.06	−1.84	−0.47
n = 9	$D$	0.05	0.01	0.10
	$K_{A}$	4.33	4.22	4.44
Kir6.2*,C166S-GFP + SUR1	$L$	0.09	−1.15	1.05
n = 6	$D$	0.70	0.29	0.91
	$K_{A}$	4.15	3.88	4.43
Kir6.2*-GFP + SUR-K205A	$L$	−0.25	−1.30	0.53
n = 9	$D$	0.18	0.06	0.32
	$K_{A}$	3.62	3.45	3.83
Kir6.2*-GFP + SUR-K205E	$L$	−0.19	−1.19	0.52
n = 9	$D$	0.30	0.13	0.47
	$K_{A}$	3.31	3.13	3.50
Negative cooperativity
Kir6.2*-GFP + SUR1	$L$	−0.42	−1.38	0.48
n = 9	$D$	0.15	0.02	0.29
	$K_{A}$	4.82	4.54	5.29
	$C$	0.17	0.06	0.36
Kir6.2*,C166S-GFP + SUR1	$L$	0.32	−0.96	1.47
n = 6	$D$	0.83	0.50	0.94
	$K_{A}$	4.43	4.04	5.14
	$C$	0.52	0.09	0.97
Kir6.2*-GFP + SUR-K205A	$L$	−0.16	−1.18	0.64
n = 9	$D$	0.52	0.32	0.64
	$K_{A}$	4.10	3.73	4.68
	$C$	0.35	0.10	0.91
Kir6.2*-GFP + SUR-K205E	$L$	−1.11	0.99
n = 9	$D$	0.58	0.32	0.73
	$K_{A}$	3.71	3.34	4.41
	$C$	0.45	0.10	0.96

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line	HEK-293T (H. sapiens)	LGC Standards (ATCC CRL-3216)
Transfected construct (Escherichia. coli)	pANAP	Addgene
Transfected construct	pcDNA4/TO	Addgene
Transfected construct (Aequorea victoria)	pCGFP_EU	Gouaux Laboratory (Vollum Institute, Portland OR USA)
Transfected construct (Homo sapiens)	peRF1-E55D	Chin Laboratory (MRC Laboratory of Molecular Biology, Cambridge UK)
Antibody	Anti-HA High Affinity; Rat monoclonal antibody (clone 3F10)	Roche	(Roche Cat# 11867423001, RRID:AB_10094468)	(1:1000)
Antibody	Peroxidase-AffiniPure Goat Anti-Rat IgG (H + L) antibody	Jackson ImmunoResearch Labs	(Jackson ImmunoResearch Labs Cat# 112-035-003, RRID:AB_2338128)	Western blots: (1:20,000) Surface expression: (1:2000)
Chemical compound, drug	trinitrophenyl-ATP (TNP-ATP)	Jena Bioscience (Jena, Germany)
Chemical compound, drug	L-3-(6-acetylnaphthalen-2-ylamino)−2-aminopropionic acid	Asis Chemicals (Waltham, MA)