Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

Abstract

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.

Fig. 1. Schematic showing the structure of the NBD of an archetypal ABC transporter. Below is the sequence alignment of the linker region of the NBDs of human SUR1, SUR2, CFTR and MDR1, showing the extensive sequence conservation.

Fig. 2. Mean K_ATP conductance recorded in the presence of 100 µM MgADP from patches excised from oocytes co-expressing Kir6.2 and either wild-type SUR1, SUR2A or SUR2B (WT, white bars), or SUR1-S1R, SUR2A-S1R or SUR2B-S1R (S1R, black bars). The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before and after exposure to MgADP (Gc). The number of patches is given above the bar.

Fig. 3. (A) Macroscopic currents recorded from oocytes co-expressing Kir6.2 and either SUR1, SUR2A, SUR2B, SUR1-S2R, SUR2A-S2R or SUR2B-S2R in response to a series of voltage ramps from –110 to +100 mV. ADP (100 µM) was added to the intracellular solution as indicated by the bars. All solutions contained Mg²⁺. (B) Mean K_ATP conductance recorded in the response to 100 µM ADP in the presence of 2 mM Mg²⁺ from patches excised from oocytes co-expressing Kir6.2 and either wild-type SUR1, SUR2A or SUR2B (WT, white bars), or SUR1-S2R, SUR2A-S2R or SUR2B-S2R (S2R, black bars). The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before and after exposure to MgADP (Gc). The number of patches is given above the bar. (C) Mean K_ATP conductance recorded in the response to 100 µM ADP in the absence of Mg²⁺ from patches excised from oocytes co-expressing Kir6.2 and either wild-type SUR1 (WT, white bar), or SUR1-S1R, SUR2A-S1R or SUR2B-S1R (S1R, grey bars), or SUR1-S2R, SUR2A-S2R or SUR2B-S2R (S2R, black bars). The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before and after exposure to ADP (Gc). The number of patches is given above the bar.

Fig. 4. (A) Mean K_ATP conductance recorded in response to 100 µM or 1 mM MgATP from patches excised from oocytes co-expressing Kir6.2-R50G and either wild-type or mutant SUR1 or SUR2B as indicated. The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before exposure to MgADP (Gc). The number of patches is given above each bar. (B) Mean relationship between K_ATP channel activation and MgADP concentration for Kir6.2-R50G/SUR1 (black squares; n = 3–5) and Kir6.2-R50G/SUR1-S1R (black triangles; n = 4–8). The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before and after exposure to MgADP (Gc). The lines are fitted to the modified Hill equation: where EC₅₀ is the MgADP concentration that gives half-maximal response and L is the maximal activation. For Kir6.2-R50G/SUR1, L = 2.4, h = 1.3 and EC₅₀ = 79 µM; for Kir6.2-R50G/SUR1-S1R, L = 1.7, h = 1.1 and EC₅₀ = 75 µM.

Fig. 5. Mean K_ATP conductance recorded from patches excised from oocytes co-expressing Kir6.2 and either wild-type SUR1 or SUR containing mutations in the linker of NBD2 (SUR1-Q1426A and SUR1-H1537A). Currents were recorded in the presence of 100 µM ADP with (white bars) or without (black bars) 2 mM Mg²⁺. The slope conductance (G) is expressed as a fraction of the mean of that obtained in control solution before and after exposure to ADP (Gc). The number of patches is given above the bar.

Fig. 6. (A) Western blotting of wild-type and mutant SUR1. Membrane proteins (25 µg) from untransfected COS-7 cells (lane 4) or cells expressing wild-type SUR1 (lane 1), SUR1-S1R (lane 2) or SUR1-S2R (lane 3) were separated by 7% SDS–PAGE. SURs were detected with anti-NBD1 antibody (Matsuo et al., 1999). Experiments were performed in duplicate. (B and C) Photoaffinity labelling of wild-type and mutant SUR1 with 8-azido-[α-³²P]ATP. Membrane proteins (25 µg) from untransfected COS-7 cells (lane 1) or cells expressing wild-type SUR1 (lanes 2–4), SUR1-S1R (lanes 5–7) or SUR1-S2R (lane 8–10) were incubated with 50 µM 8-azido-[α-³²P]ATP, in the absence of other nucleotide (lanes 1, 2, 5 and 8) or in the presence of 100 µm ADP (lanes 3, 6 and 9) or 100 µm ATP (lanes 4, 7 and 10), for 10 min on ice and then UV irradiated. Photoaffinity-labelled proteins were subject to mild trypsin digestion. The tryptic fragments were immunoprecipitated with anti-NBD1 (Figure 6B) or anti-NBD2 (Figure 6C) antibody and separated by 12% PAGE. A 65 kDa fragment containing NBD2 and a 35 kDa fragment containing NBD1 are indicated. Note that NBD2 was co-immunoprecipitated with NBD1. (D) Photoaffinity labelling of wild-type and mutant SUR1 with 8-azido-[α-³²P]ATP or 8-azido-[γ-³²P]ATP. Membrane proteins (20 µg) from cells expressing wild-type SUR1 (lanes 1, 4, 7 and 10), SUR1-S1R (lanes 2, 5, 8 and 11) or SUR1-S2R (lane 3, 6, 9 and 12) were incubated with 50 µM 8-azido-[α-³²P]ATP (lanes 1–6) or 8-azido-[γ-³²P]ATP (lanes 7–12) for 10 min at 37°C and then UV irradiated. Photoaffinity-labelled proteins were subjected to mild trypsin digestion. The tryptic fragments were immunoprecipitated with anti-NBD1 (lanes 1–3 and 7–9) or anti-NBD2 (lanes 4–6 and 10–12) antibody and separated by 12% PAGE. A 65 kDa fragment containing NBD2 and a 35 kDa fragment containing NBD1 are indicated. Note that NBD2 was co-immunoprecipitated with NBD1.

Fig. 7. NBD1 of SURx binds ATP or ADP, and has little or no ATPase activity (Bienengraeber et al., 2000; Matsuo et al., 2000). NBD2 of SURx binds MgATP or MgADP depending on their ratio and concentration. Bound MgATP is hydrolysed to MgADP. MgADP activates the K_ATP channel via NBD2. The S2R mutation of SUR1 and SUR2B abolishes channel activation by Mg-nucleotides completely. In contrast, the S1R mutation in SURx reduces channel activation by MgADP or MgATP, possibly by influencing transduction at NBD2. The S2R mutation of SUR2A does not alter K_ATP channel activation by MgADP, suggesting that the SUR2A tail prevents (or compensates for) the effect of the S2R mutation on signal transduction.