Trapp S et al. (1998), Molecular analysis of ATP-sensitive K channel g...

XB-ART-14350

J Gen Physiol 1998 Sep 01;1123:333-49. doi: 10.1085/jgp.112.3.333.

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Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.

Trapp S , Proks P , Tucker SJ , Ashcroft FM .

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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.

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Species referenced: Xenopus laevis
Genes referenced: abcc8 dtl kcnj11

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	Figure 9. A simple kinetic scheme for Kir6.2ΔC26 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 wtKir6.2ΔC26 and Kir6.2ΔC26-C166S macroscopic currents. (A) Macroscopic currents recorded from cell-attached patches on oocytes injected with mRNA encoding wtKir6.2ΔC26 (a) or Kir6.2ΔC26-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 −110 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 wtKir6.2ΔC26 (wt) or Kir6.2ΔC26-C166S (C166S). The number of oocytes is given above the bars. (D) Macroscopic current–voltage relations recorded in response to a voltage ramp from −110 to +100 mV from inside-out patches on oocytes injected with mRNA encoding wtKir6.2ΔC26 (a) or Kir6.2ΔC26-C166S (b).
	Figure 2. Effects of ATP on wtKir6.2ΔC26 and Kir6.2ΔC26-C166S macroscopic currents. (A) Macroscopic currents recorded from an inside-out patch on an oocyte injected with mRNA encoding Kir6.2ΔC26-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 −110 to +100 mV. The patch was excised at the point indicated by the arrow. The dashed line indicates the zero current level. (B) Macroscopic wtKir6.2ΔC26 (i) or Kir6.2ΔC26-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 −110 to +100 mV. The dashed line indicates the zero current level. (C) Mean ATP dose–response relationships for wtKir6.2ΔC26 currents (○, n = 7) and Kir6.2ΔC26-C166S currents (•, 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 −20 and −100 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 Kir6.2ΔC26-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 wtKir6.2ΔC26 and Kir6.2ΔC26-C166S single-channel currents. Single-channel currents at −60 mV (A) and mean single-channel current–voltage relations (B) recorded for wtKir6.2ΔC26 (○, n = 3) or Kir6.2ΔC26-C166S (•, n = 3) currents in inside-out patches. (C) Voltage dependence of the mean open probability of wtKir6.2ΔC26 (○, n = 3) and Kir6.2ΔC26-C166S (•, n = 3) channels.
	Figure 5. Effect of ATP on the kinetics of wtKir6.2ΔC26 and Kir6.2ΔC26-C166S single-channel currents. Single-channel currents recorded from an inside-out patch at −60 mV in the presence and absence of ATP, as indicated, from oocytes expressing Kir6.2ΔC26 (B) or Kir6.2ΔC26-C166S (A).
	Figure 6. Effects of different amino acid substitutions at position 166 of Kir6.2ΔC26 on single-channel currents and ATP sensitivity. (A) Single-channel currents recorded at −60 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 Kir6.2ΔC26-C166L, recorded from an inside-out patch at −60 mV. (C) Mean ATP dose–response relationships for wtKir6.2ΔC26 (•, n = 7) and for Kir6.2ΔC26 containing the mutations C166V (▴, n = 8), C166L (▾, n = 6), and C166M (♦, 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 −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 (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 −110 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 Kir6.2ΔC26-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 Kir6.2ΔC26 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, k−1, k2, and k−2 in the absence of ATP, calculated for Kir6.2ΔC26-C166S currents (n = 3). The rate constants were calculated from measured values of τo, τC1, 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.

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