Blocking Kir6.2 channels with SpTx1 potentiates glucose-stimulated insulin secretion from murine pancreatic β cells and lowers blood glucose in diabetic mice
- Department of Physiology, Perelman School of Medicine University of Pennsylvania, United States
Figures
Figure 1
SpTx1 does not affect the blood glucose level or potentiate glucose-stimulated insulin secretion (GSIS) from isolated pancreatic islets of wild-type B6J mice.
(A, B) Blood glucose levels (mean ± standard error of the mean [SEM], n = 5 independent experiments for each dataset) of overnight-fasted mice (8–12 weeks of age) measured at indicated time points …
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Figure 1—source data 1
Related to Figure 1A–C.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig1-data1-v1.xlsx
Figure 2
SpTx1 does not depolarize the membrane potential (Vm) of β cells in isolated pancreatic islets from wild-type mice.
(A–C) Vm traces recorded in the perforated whole-cell mode from individual β cells near the surface of isolated but intact islets from B6J wild-type mice (8–12 weeks of age). The switching of the …
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Figure 2—source data 1
Related to Figure 2A–F.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
SpTx1 inhibits hKir6.2 and mKir6.2 with markedly different affinities.
Currents through hKir6.2 coexpressed with hSUR1 (hKATP; A), mKir6.2 with mSUR1 (mKATP; B), hKir6.2 with mSUR1 (C), and mKir6.2 with hSUR1 (D) activated by adding 3 mM azide to the 100 mM K+-containin…
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Figure 3—source data 1
Related to Figure 3A–F.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
SpTx1 affinities of Kir6.2 and of its variants in the study.
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Figure 3—figure supplement 1—source data 1
Related to Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig3-figsupp1-data1-v1.xlsx
Figure 4
Residue E108 underlies the high affinity of hKir6.2 for SpTx1.
(A) Shown at the top is a comparison of partial amino acid sequences of the extracellular vestibule of the pore between the first and second transmembrane segments in hKir6.2 and mKir6.2. The three …
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Figure 4—source data 1
Related to Figure 4A–C.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig4-data1-v1.xlsx
Figure 5
A single-point mutation confers high SpTx1 affinity on mKir6.2bgd and switches the affinities of hKATP and mKATP channels for SpTx1.
Currents of mKir6.2bgd (A), mKir6.2bgd containing the triple mutations G103S, N107A, and V108E (B), mKir6.2bgd containing the single mutation V108E (C), azide-activated mKir6.2-V108E coexpressed …
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Figure 5—source data 1
Related to Figure 5A–G.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
SpTx1 depolarizes the membrane potential (Vm) of β cells in isolated pancreatic islets from EndomKir6.2V108E mice.
(A–C) Vm traces recorded in the perforated whole-cell mode from individual β cells near the surface of isolated intact islets from EndomKir6.2V108E mice (8–12 weeks of age). The switching of the …
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Figure 6—source data 1
Related to Figure 6A–F.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
DNA sequencing result and analysis of EndomKir6.2V108E mice.
(A) Chromatogram showing the sequence of a 42-nucleotide segment within the PCR-amplified products of the mKir6.2 gene in the endogenous locus; the engineered-in restriction enzyme (BanII) cleave …
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Figure 6—figure supplement 1—source data 1
DNA sequencing chromatogram containing the proximal region of the endogenous Kir6.2 gene in EndomKir6.2V108E mice related to Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
DNA sequencing chromatogram containing the distal region of the endogenous Kir6.2 gene in EndomKir6.2V108E mice related to Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig6-figsupp1-data2-v1.zip
Figure 7
SpTx1 potentiates glucose-stimulated insulin secretion (GSIS) from isolated pancreatic islets of EndomKir6.2V108E mice and counteracts the effect of diazoxide on GSIS.
(A–D) The amount of insulin per ml perifusion solution released from ~180 isolated islets of EndomKir6.2V108E mice (8–12 weeks of age) as a percentage of the total insulin content is plotted against …
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Figure 7—source data 1
Related to Figure 7A–E.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig7-data1-v1.xlsx
Figure 8 with 2 supplements
SpTx1 triggers a transient rise of plasma insulin and lowers the elevated blood glucose level in diabetic Rosa26mKir6.2NDM-V108E mice.
Blood glucose (A, B) and corresponding plasma insulin (C, D) levels (mean ± standard error of the mean [SEM]) of overnight-fasted mice (8–12 weeks of age) at indicated time points during a 2-hr …
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Figure 8—source data 1
Related to Figure 8A–D.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
DNA sequencing results and analysis of Rosa26mKir6.2NDM-V108E mice.
(A) Chromatogram showing the sequences of a 42-nucleotide segment within the PCR-amplified products of the mKir6.2 gene in the endogenous (A) or Rosa26 (B) locus; the engineered-in restriction …
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Figure 8—figure supplement 1—source data 1
DNA sequencing chromatogram containing the proximal region of the endogenous Kir6.2 gene in Rosa26mKir6.2NDM-V108E mice related to Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp1-data1-v1.zip
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Figure 8—figure supplement 1—source data 2
DNA sequencing chromatogram containing the distal region of the endogenous Kir6.2 gene in Rosa26mKir6.2NDM-V108E mice related to Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp1-data2-v1.zip
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Figure 8—figure supplement 1—source data 3
DNA sequencing chromatogram containing the proximal region of the NDM-causing mutant Kir6.2 transgene in Rosa26mKir6.2NDM-V108E mice related to Figure 8—figure supplement 1B.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp1-data3-v1.zip
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Figure 8—figure supplement 1—source data 4
DNA sequencing chromatogram containing the mid region of the NDM-causing mutant Kir6.2 transgene in Rosa26mKir6.2NDM-V108E mice related to Figure 8—figure supplement 1B.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp1-data4-v1.zip
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Figure 8—figure supplement 1—source data 5
DNA sequencing chromatogram containing the distal region of the NDM-causing mutant Kir6.2 transgene in Rosa26mKir6.2NDM-V108E mice related to Figure 8—figure supplement 1B.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp1-data5-v1.zip
Figure 8—figure supplement 2
The N107Y mutation does not reduce the SpTx1 sensitivity of hKir6.2bgd.
Fractions of remaining channel currents (I/Io) of hKir6.2bgd (open squares) or hKir6.2bgd-N107Y (filled squares) plotted against the concentration of SpTx1. The curves superimposed onto the data …
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Figure 8—figure supplement 2—source data 1
The N107Y mutation does not alter SpTx1 sensitivity of hKir6.2 related to Figure 8—figure supplement 2.
- https://cdn.elifesciences.org/articles/77026/elife-77026-fig8-figsupp2-data1-v1.xlsx
