Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor

Abstract

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

Figure 1

Diazoxide stimulation of recombinant K_ATP channels requires hydrolyzable nucleotides. Representative currents (n = 4–5 patches) recorded from inside-out membrane patches containing wild-type K_ATP channels at −50 mV. Inward currents are shown as upward deflections. Patches were exposed to differing [ATP], [ADP], [diazoxide], and [Mg²⁺] as indicated by the bars above the records.

Figure 2

Diazoxide stimulation is reduced or abolished by NBF2 mutations. Representative currents (n = 3–5 patches) recorded from inside-out membrane patches containing wild-type or NBF2 mutant K_ATP channels (as indicated) at −50 mV. Patches were exposed to differing [ATP] and [diazoxide], as indicated by the bars above the records. Free [Mg²⁺] was maintained at 1 mM in all ATP-containing solutions.

Figure 4

Currents (see text) in 0.1 mM ATP solutions, relative to current in zero ATP solution, with (filled columns) and without (open columns) (A) 0.3 mM diazoxide, or (B) 0.5 mM ADP. Bars indicate mean ± SEM for n = 3–6 patches in each case. n.d., not done. Free Mg²⁺ was maintained at 1 mM in all ATP-containing solutions.

Figure 3

Kinetics of diazoxide stimulation are altered by NBF1 mutations. Representative currents (n = 3–6 patches) recorded from inside-out membrane patches containing wild-type or NBF1 mutant K_ATP channels (as indicated) at −50 mV. Patches were exposed to differing [ATP] and [diazoxide], as indicated by the bars above the records. Free [Mg²⁺] was maintained at 1 mM in all ATP-containing solutions.

Figure 5

Time constants of activation (τ_ACT) and deactivation (τ_DEACT) during and after stimulation by (A) diazoxide or (B) ADP for wild-type (WT) and various mutant K_ATP channels. Bars indicate mean ± SEM for n = 3–6 patches in each case. (C) Currents (see text) in 0.1 mM ATP solution relative to current in zero ATP solution, with (•) and without (○, control) diazoxide, and time-constants of activation (τ_ACT) during stimulation by diazoxide, versus the test diazoxide concentration, for G827R mutant channels. Free Mg²⁺ was maintained at 1 mM in all ATP-containing solutions.

Figure 6

ADP stimulation is abolished by NBF2 mutations, and the kinetics are altered by NBF1 mutations. Representative currents recorded from inside-out membrane patches (n = 4–7) containing wild-type or mutant K_ATP channels (G827R and Q834H in NBF1, G1485D in NBF2) at −50 mV. Patches were exposed to differing [ATP] and [ADP], as indicated by the bars above the records. Free [Mg²⁺] was maintained at 1 mM in all ATP-containing solutions.

Figure 7

Sensitivity to ATP in the absence of Mg²⁺ is unaltered by NBF1 or NBF2 mutations. Currents in 0.1 or 0.01 mM ATP solutions relative to current in zero ATP solution for wild-type (WT) and mutant channels as indicated. Bars indicate mean ± SEM for n = 4–5 patches in each case.

Figure 8

Model to explain nucleotide regulation of K_ATP channels. The SUR1 subunit acts as a ‘hypersensitivity switch' to modulate ATP sensitivity. ATP⁴⁻ binds to either the Kir6.2 subunit itself, or a third, unknown protein to close K_ATP channels with an intrinsic K _i of ∼100 μM (Tucker et al., 1997). When coupled to the SUR1 subunit, SUR1 exerts a hypersensitizing effect on channel activity to reduce the K _i to ∼10 μM (Inagaki et al., 1995). This hypersensitizing switch is turned off by nucleotide hydrolysis at the SUR1 NBFs, and this effect is mimicked or enhanced by MgADP or diazoxide. Alternatively, the hypersensitizing effect is abolished when the channels are treated with trypsin, such that MgADP and diazoxide can no longer stimulate channel activity.

Figure 9

Trypsin treatment abolishes ADP stimulation of wild-type channels and shifts ATP sensitivity in wild-type and NBF mutant channels. Representative currents recorded from inside-out membrane patches containing wild-type (n = 3) or G1485D (n = 4) mutant K_ATP channels (as indicated) at −50 mV. Patches were exposed to differing [ATP] and [ADP], as indicated by the bars above the records. Each record is cut (dashed line) for ∼60 s during application of trypsin at 1 mg/ml, as indicated. Free [Mg²⁺] was maintained at 1 mM in all ATP-containing solutions.

Figure 10

Pyrophosphate and vanadate do not activate K_ATP channels. Representative currents (n = 3 in each case) recorded from inside-out membrane patches containing wild-type K_ATP channels at −50 mV. Patches were exposed to differing [ATP], as indicated by the bars above the record. 2 mM pyrophosphate (PPi) or 1 mM vanadate (VO₄^{3 −}) were added where indicated. Free [Mg²⁺] was maintained at 1 mM in all ATP-containing solutions.