Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Gen Physiol
1998 Sep 01;1123:333-49. doi: 10.1085/jgp.112.3.333.
Show Gene links
Show Anatomy links
Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.
Trapp S
,
Proks P
,
Tucker SJ
,
Ashcroft FM
.
???displayArticle.abstract???
The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.
???displayArticle.pubmedLink???
9725893
???displayArticle.pmcLink???PMC2229413 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 9. A simple kinetic scheme for Kir6.2ÎC26 channel gating in the presence of ATP. C2(ATP) and C3(ATP) indicate additional closed states observed in the presence of ATP. The dotted lines represent the rate constants affected by the C166S mutation.
Figure 1. Properties of wtKir6.2ÎC26 and Kir6.2ÎC26-C166S macroscopic currents. (A) Macroscopic currents recorded from cell-attached patches on oocytes injected with mRNA encoding wtKir6.2ÎC26 (a) or Kir6.2ÎC26-C166S (b). The patch was excised at the point indicated by the arrow. Currents were elicited in response to a series of voltage ramps from â110 to +100 mV. The dashed line indicates the zero current level. (B) Putative membrane topology of Kir6.2 with the position of C166 marked. (C) Mean current amplitude recorded before and after excision of membrane patches from oocytes expressing wtKir6.2ÎC26 (wt) or Kir6.2ÎC26-C166S (C166S). The number of oocytes is given above the bars. (D) Macroscopic currentâvoltage relations recorded in response to a voltage ramp from â110 to +100 mV from inside-out patches on oocytes injected with mRNA encoding wtKir6.2ÎC26 (a) or Kir6.2ÎC26-C166S (b).
Figure 2. Effects of ATP on wtKir6.2ÎC26 and Kir6.2ÎC26-C166S macroscopic currents. (A) Macroscopic currents recorded from an inside-out patch on an oocyte injected with mRNA encoding Kir6.2ÎC26-C166S. ATP was added to the internal solution as indicated by the bar. Currents were elicited in response to a series of voltage ramps from â110 to +100 mV. The patch was excised at the point indicated by the arrow. The dashed line indicates the zero current level. (B) Macroscopic wtKir6.2ÎC26 (i) or Kir6.2ÎC26-C166S (ii) currents. ATP was added to the internal solution as indicated by the bar. Currents were elicited in response to a series of voltage ramps from â110 to +100 mV. The dashed line indicates the zero current level. (C) Mean ATP doseâresponse relationships for wtKir6.2ÎC26 currents (â, n = 7) and Kir6.2ÎC26-C166S currents (â¢, n = 10). Test solutions were alternated with control solutions and the slope conductance (G) is expressed as a fraction of the mean (Gc) of that obtained in control solution before and after exposure to ATP. Conductance was measured between â20 and â100 mV and is the mean of five voltage ramps. The dotted and solid lines are the best fit of the data to the Hill equation (Eq. 1) using the mean values for Ki and h given in the text. The dashed line has been fit to the Kir6.2ÎC26-C166S data using a modified form of the Hill equation (Eq. 2) in which it is not assumed that ATP block at saturating concentrations is complete. Mean values for Ki and h are given in the text.
Figure 3. Properties of wtKir6.2ÎC26 and Kir6.2ÎC26-C166S single-channel currents. Single-channel currents at â60 mV (A) and mean single-channel currentâvoltage relations (B) recorded for wtKir6.2ÎC26 (â, n = 3) or Kir6.2ÎC26-C166S (â¢, n = 3) currents in inside-out patches. (C) Voltage dependence of the mean open probability of wtKir6.2ÎC26 (â, n = 3) and Kir6.2ÎC26-C166S (â¢, n = 3) channels.
Figure 5. Effect of ATP on the kinetics of wtKir6.2ÎC26 and Kir6.2ÎC26-C166S single-channel currents. Single-channel currents recorded from an inside-out patch at â60 mV in the presence and absence of ATP, as indicated, from oocytes expressing Kir6.2ÎC26 (B) or Kir6.2ÎC26-C166S (A).
Figure 6. Effects of different amino acid substitutions at position 166 of Kir6.2ÎC26 on single-channel currents and ATP sensitivity. (A) Single-channel currents recorded at â60 mV from inside-out patches excised from oocytes injected with the different mutant mRNAs indicated. (B) Three different types of single-channel kinetics observed for Kir6.2ÎC26-C166L, recorded from an inside-out patch at â60 mV. (C) Mean ATP doseâresponse relationships for wtKir6.2ÎC26 (â¢, n = 7) and for Kir6.2ÎC26 containing the mutations C166V (â´, n = 8), C166L (â¾, n = 6), and C166M (â¦, n = 7), measured for macroscopic currents recorded from giant inside-out membrane patches. Test solutions were alternated with control solutions and the slope conductance (G) is expressed as a fraction of the mean (Gc) of that obtained in control solution before and after exposure to ATP. Conductance was measured between â20 and â100 mV and is the mean of five voltage ramps. The lines are the best fit of the data to the Hill equation (Eq. 1) using the mean values for Ki and h given in Table I.
Figure 7. Effect of mutations at residue C166 on the coupling of Kir6.2 to SUR1. (A and B) Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from â110 to +100 mV. Oocytes were injected with mRNAs encoding Kir6.2 + SUR1 (A) or Kir6.2-C166S + SUR1 (B). MgADP, MgATP, diazoxide (DZ), or tolbutamide (TB) were added to the internal solution as indicated by the bars. (C) Mean macroscopic slope conductance recorded in the presence of the agents indicated (G), expressed as a fraction of the mean (Gc) of that obtained in control solution (no additions) before and after exposure to the test compound(s). The dashed line indicates the current level in the absence of the test compound. Oocytes were injected with mRNAs encoding Kir6.2 + SUR1 (white bars), Kir6.2-C166S + SUR1 (black bars), or Kir6.2ÎC26-C166V + SUR1 (hatched bars). The number of oocytes is given above the bars. *P < 0.05, **P < 0.01 against Kir6.2 + SUR1.
Figure 8. (A) A simple kinetic scheme for Kir6.2ÎC26 channel gating in ATP-free solution, where O is the open state, C1 represents the short closed state observed within a burst of openings, and C2 represents the long closed state that governs the interburst duration. The dotted line represents the rate constant affected by the C166S mutation. (B) Voltage dependence of the rate constants k1, kâ1, k2, and kâ2 in the absence of ATP, calculated for Kir6.2ÎC26-C166S currents (n = 3). The rate constants were calculated from measured values of Ïo, ÏC1, Po, and percent C2. The lines are fitted to the equation k = a + ko exp (zδVF/RT), where ko is the rate constant at a membrane potential of 0 mV, V is the membrane potential, zδ is a term describing the electrical distance through the membrane, and a is a voltage-independent component. (C) Values of the parameters used to fit the voltage dependence of the rate constants shown in B.
Aguilar-Bryan,
Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion.
1995, Pubmed
Aguilar-Bryan,
Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion.
1995,
Pubmed
Ashcroft,
Properties of single potassium channels modulated by glucose in rat pancreatic beta-cells.
1988,
Pubmed
Clement,
Association and stoichiometry of K(ATP) channel subunits.
1997,
Pubmed
Dunne,
Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells.
1989,
Pubmed
Findlay,
ATP maintains ATP-inhibited K+ channels in an operational state.
1986,
Pubmed
Gillis,
Effects of sulfonamides on a metabolite-regulated ATPi-sensitive K+ channel in rat pancreatic B-cells.
1989,
Pubmed
Gribble,
MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit.
1998,
Pubmed
,
Xenbase
Gribble,
The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.
1997,
Pubmed
,
Xenbase
Gribble,
Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
Holmgren,
Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating.
1997,
Pubmed
Inagaki,
Subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ channel.
1997,
Pubmed
Inagaki,
Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor.
1995,
Pubmed
Inagaki,
A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels.
1996,
Pubmed
Jackson,
Successive openings of the same acetylcholine receptor channel are correlated in open time.
1983,
Pubmed
Kakei,
Properties of adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells.
1985,
Pubmed
Kozlowski,
Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line.
1989,
Pubmed
Kyte,
A simple method for displaying the hydropathic character of a protein.
1982,
Pubmed
Liu,
Gated access to the pore of a voltage-dependent K+ channel.
1997,
Pubmed
Lopatin,
Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification.
1994,
Pubmed
,
Xenbase
Loukin,
Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.
1997,
Pubmed
,
Xenbase
Nichols,
ATP dependence of KATP channel kinetics in isolated membrane patches from rat ventricle.
1991,
Pubmed
Nichols,
Adenosine diphosphate as an intracellular regulator of insulin secretion.
1996,
Pubmed
Ohno-Shosaku,
Dual effects of ATP on K+ currents of mouse pancreatic beta-cells.
1987,
Pubmed
Proks,
Phentolamine block of KATP channels is mediated by Kir6.2.
1997,
Pubmed
,
Xenbase
Sakura,
Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle.
1995,
Pubmed
Shyng,
Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.
1997,
Pubmed
Shyng,
Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit.
1997,
Pubmed
Shyng,
Octameric stoichiometry of the KATP channel complex.
1997,
Pubmed
Trapp,
Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits.
1997,
Pubmed
,
Xenbase
Trapp,
Mechanism of ATP-sensitive K channel inhibition by sulfhydryl modification.
1998,
Pubmed
,
Xenbase
Tucker,
Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor.
1997,
Pubmed
,
Xenbase
Tucker,
Molecular determinants of KATP channel inhibition by ATP.
1998,
Pubmed
,
Xenbase
Zilberter,
Gating kinetics of ATP-sensitive single potassium channels in myocardial cells depends on electromotive force.
1988,
Pubmed
Zong,
Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.
1998,
Pubmed