scholarly journals Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

2019 ◽  
Author(s):  
Samuel G. Usher ◽  
Frances M. Ashcroft ◽  
Michael C. Puljung

AbstractPancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Samuel G Usher ◽  
Frances M Ashcroft ◽  
Michael C Puljung

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.


1990 ◽  
Vol 240 (1298) ◽  
pp. 397-410 ◽  

ATP-sensitive K + -channel currents were recorded from isolated mem­brane patches and voltage-clamped CRI-G1 insulin-secreting cells. Inter­nal Mg 2+ ions inhibited ATP-K + channels by a voltage-dependent block of the channel current and decrease of open-state probability. The run­ down of ATP-K + channel activity was also shown to be [Mg 2+ ] i depen­dent, being almost abolished in Mg 2+ -free conditions. Substitution of Mn 2+ for Mg 2+ did not prevent run-down, nor did the presence of phos­phate-donating nucleotides, a protease or phosphatase inhibitor or replacement of Cl by gluconate.


2007 ◽  
Vol 178 (7) ◽  
pp. i17-i17
Author(s):  
Decha Enkvetchakul ◽  
Iana Jeliazkova ◽  
Jaya Bhattacharyya ◽  
Colin G. Nichols

2012 ◽  
Vol 140 (5) ◽  
pp. 529-540 ◽  
Author(s):  
Lei Yang ◽  
Johan Edvinsson ◽  
Lawrence G. Palmer

We investigated the effects of changing extracellular K+ concentrations on block of the weak inward-rectifier K+ channel Kir1.1b (ROMK2) by the three intracellular cations Mg2+, Na+, and TEA+. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K+ in the inside (cytoplasmic) solution and 11 mM K+ in the outside (extracellular) solution, these three cations blocked K+ currents with a range of apparent affinities (Ki (0) = 1.6 mM for Mg2+, 160 mM for Na+, and 1.8 mM for TEA+) but with similar voltage dependence (zδ = 0.58 for Mg2+, 0.71 for Na+, and 0.61 for TEA+) despite having different valences. When external K+ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg2+ but not for Na+ or TEA+. In contrast, the N152Y mutation increased the affinity for block by TEA+ but not for Na+ or Mg2+. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg2+ but had a small effect on that by Na+. TEA+ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K+ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K+ are explained by shifts in the occupancy of sites within the selectivity filter by K+ ions.


2011 ◽  
Vol 69 (2) ◽  
pp. 241-251 ◽  
Author(s):  
Cornelia Eisenach ◽  
Zhong‐Hua Chen ◽  
Christopher Grefen ◽  
Michael R. Blatt

2000 ◽  
Vol 20 (22) ◽  
pp. 8298-8304 ◽  
Author(s):  
Jiyeon Kwak ◽  
Myeong Hyeon Wang ◽  
Sun Wook Hwang ◽  
Tae-Yoon Kim ◽  
Soon-Youl Lee ◽  
...  

1995 ◽  
Vol 80 (5) ◽  
pp. 825-834 ◽  
Author(s):  
HL Reeve ◽  
EK Weir ◽  
DP Nelson ◽  
DA Peterson ◽  
SL Archer

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