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J Gen Physiol
1998 Sep 01;1123:325-32. doi: 10.1085/jgp.112.3.325.
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Mechanism of ATP-sensitive K channel inhibition by sulfhydryl modification.
Trapp S
,
Tucker SJ
,
Ashcroft FM
.
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ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned beta cell KATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the KATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.
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9725892
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Figure 2. Effects of pCMPS on Kir6.2ΔC26 currents. Macroscopic currents recorded from an inside-out patch excised from an oocyte injected with Kir6.2ΔC26 mRNA in response to voltage ramps from −110 to +100 mV. pCMPS and DTT were added to the internal solution as indicated by the bars.
Figure 3. Mutation of C42 abolishes pCMPS but not ATP block of KATP currents. (A) Putative membrane topology of Kir6.2 with the location of intracellular cysteine residues marked. (B and C) Macroscopic currents recorded from inside-out patches excised from oocytes coinjected with either Kir6.2ΔC26-C42V and SUR1 mRNAs (B) or Kir6.2-C42A and SUR1 mRNAs (C). Currents were recorded in response to voltage ramps from −110 to +100 mV. pCMPS and ATP were added to the internal solution as indicated by the bars.
Figure 4. Effect of cysteine mutations on the rate of pCMPS block. (A and B) Macroscopic currents recorded at −100 mV from inside-out patches excised from oocytes injected with Kir6.2ΔC26 or Kir6.2ΔC26-C166S mRNA. pCMPS was added to the internal solution as indicated by the bars. The dotted line indicates the zero current level and the dashed line is an extrapolated linear fit to the slope of the rundown in control solution and extrapolated. (C) Mean time constant for pCMPS block of wild-type and mutant Kir6.2ΔC26 channels (open bars), and for Kir6.2 or Kir6.2ΔC26-C197V coexpressed with SUR1 (solid bars). The number of oocytes is given above the bars. *P = 0.0005 against wild-type Kir6.2ΔC26 currents (wt). +P < 0.005 against wild-type (Kir6.2/SUR1) currents.
Figure 5. (A) Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from −110 to +100 mV from oocytes injected with mRNAs encoding SUR1 and either Kir6.2ΔC26 or Kir6.2ΔC26-C42V. ATP was added to the internal solution as indicated by the bar. (B) Mean ATP dose– response relationships for Kir6.2ΔC26/SUR1 currents (○, n = 5) and Kir6.2ΔC26-C42V/SUR1 currents (•, n = 4). Test solutions were alternated with control solutions and the slope conductance (G) is expressed as a percentage of the mean (Gc) of that obtained in control solution before and after exposure to ATP. Conductance was measured between −20 and −100 mV and is the mean of five voltage ramps. The lines are the best fit of the data to the Hill equation using the mean values for Ki and h given in the text. (C) Mean current amplitude recorded during exposure to ATP, expressed as a fraction of the mean current amplitude before and after exposure to ATP for patches excised from oocytes injected with wild-type or mutant Kir6.2ΔC26 mRNAs (top), or wild-type or mutant Kir6.2 together with SUR1 (bottom). The dashed line indicates the control current level (in the absence of ATP). The number of oocytes is given above the bars.
Figure 6. Coupling to SUR1 is reduced when C42 in Kir6.2 is mutated to alanine or valine. (A and B) Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from −110 to +100 mV from oocytes injected with mRNAs encoding SUR1 and either wtKir6.2 or Kir6.2-C42A. MgADP, ATP, 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 MgADP, diazoxide, or tolbutamide, expressed as a percentage of the slope conductance in control solution (no additions), for the indicated channels. Diazoxide was added in the presence of MgATP. The dashed line indicates the current level in control solution. The number of oocytes is given above the bars. *P < 0.01; +P < 0.05.
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