Potassium channels selectively conduct K+ ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba2+ as a probe to determine the location of the ligand‐sensitive gate in an inwardly rectifying K+ channel (Kir6.2). To a K+ channel, Ba2+ and K+ are of similar sizes, but Ba2+ blocks the pore by binding within the selectivity filter. We found that internal Ba2+ could still access its binding site when the channel was shut, which indicates that the ligand‐sensitive gate lies above the Ba2+‐block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage‐dependent gate of KV channels, which is located at the intracellular mouth of the pore.
Ion channels are intrinsic membrane proteins that act as gated pores to regulate the movement of ions across cell membranes. Their opening and closing (gating) is central to their function and is regulated by membrane voltage, mechanical force or ligand binding (Hille, 2001). Potassium channels are one of the largest groups of ion channels, and have pivotal roles in the function of many cell types. Mutations in K+‐channel genes, many of which impair gating, result in diseases such as epilepsy, deafness, cardiac disorders and impaired insulin secretion (Ashcroft, 2000).
The crystal structure of the bacterial K+ channel KcsA has provided a paradigm for the pore of K+ channels (Doyle et al., 1998; Zhou et al., 2001). Each of the four subunits of KcsA has two transmembrane helices, which form an inverted teepee (Fig. 1A). The helix‐bundle crossing region defines the inner entrance of the pore and is proposed to serve as an intracellular gate to the channel (Fig. 1B, b; Doyle et al., 1998; Liu et al., 2001; Perozo et al., 1999). Support for this idea comes from the fact that the crystal structure of the bacterial K+ channel MthK, imaged in the open state, reveals a widening of the pore at the helix‐bundle crossing region (Jiang et al., 2002). Functional studies indicate that the activation gate of the voltage‐dependent K+ (KV) channel Shaker is also located at the bundle crossing (Del Camino et al., 2000; Del Camino & Yellen, 2001). Access of the small cations Cd+ and Ag+ (and by inference K+) to the internal cavity is regulated by this gate (Del Camino & Yellen, 2001).
However, evidence is accumulating that the selectivity filter of K+ channels might also act as a gate (Fig. 1A, B, a). At low K+, the selectivity filter of KcsA is occluded by the rotation of Gly 77 into the permeation path (Zhou et al., 2001) and spin‐labelling studies suggest that small conformational changes take place at the selectivity filter during gating (Perozo et al., 1999). The ability of permeant ions to influence KV channel gating (Armstrong et al., 1982) is also most readily explained by an effect on the selectivity filter. This has led to the suggestion that the selectivity filter of KV channels might contribute to intrinsic gating, even if it does not serve as the voltage‐sensitive gate. In the case of cyclic‐nucleotide‐gated channels, a recent study suggests that the ligand‐sensitive gate lies above the helix‐bundle crossing and thus, by default, might reside within the selectivity filter (Flynn & Zagotta, 2001). The location of the ligand‐sensitive gate of the inwardly rectifying K+ (Kir) channels is unknown. Here, by studying block by internal Ba2+, we provide direct evidence that the ligand‐sensitive gate of the Kir channel Kir6.2 is not located at the helix‐bundle crossing but lies within, or above, the selectivity filter.
Like KcsA, Kir6.2 has two transmembrane helices, linked by a pore loop, and cytoplasmic amino and carboxy termini (Fig. 1A). Four pore‐forming Kir6.2 subunits associate with four regulatory SUR subunits to form the ATP‐sensitive K+ channel, and binding of ATP to the cytosolic domains of Kir6.2 results in channel closure (Ashcroft & Gribble, 1999). In the absence of ATP, KATP channels exhibit spontaneous bursts of openings, separated by long closed intervals (Trapp et al., 1998). ATP decreases the frequency and duration of the bursts and increases the duration and frequency of the long closed times. Mutations in the helix‐bundle crossing region dramatically increase the burst duration, but do not alter the duration of the long closed times (Trapp et al., 1998). This suggests that the helix‐bundle crossing does not serve as the ligand‐sensitive gate.
We exploited the fact that Ba2+ binds within the selectivity filter of K+ channels (Neyton & Miller, 1988a,b; Jiang & MacKinnon, 2000) to determine the location of the gate closed by ATP. If the helix‐bundle crossing serves as the ligand‐sensitive gate, then ATP should slow the rate and reduce the affinity of the internal Ba2+ block. In contrast, if the gate lies above the Ba2+‐binding site, the Ba2+ block should be largely unaffected by the presence of the nucleotide.
Fig. 2A shows that Kir6.2/SUR1 currents were unaffected by the presence of intracellular Ba2+ at −40 mV, but that the cation induced a rapid time‐ and voltage‐dependent block of outward currents on depolarization. Increasing ATP to 15 μM, which decreased Kir6.2/SUR1 currents by ∼70% (Fig. 2C), produced a small but significant acceleration of block by Ba2+ (Fig. 2B, D). This effect is the opposite of that expected if the access of Ba2+ to its binding site is impaired when the channel is shut by ATP, and implies that the gate lies above the Ba2+‐binding site. Ba2+ did not alter the half‐maximal inhibitory concentration (IC50) for ATP inhibition (6 ± 2 μM (n = 6) compared with 6 μM in the absence of Ba2+ (Gribble et al., 1998)), which argues against an electrostatic (gating‐independent) effect of ATP on Ba2+ binding.
To explain how ATP accelerates block by Ba2+, we simulated the block of macroscopic currents, using rate constants obtained from single‐channel recordings (Fig. 3A). Regardless of the free parameters used to fit the kinetic model, these simulations show that if Ba2+ binds only to the open state (that is, above the gate), a decrease in channel open probability (Po) reduces the rate and extent of block (Fig. 3B, C). Because this is not observed experimentally, the gate closed by ATP cannot lie below the Ba2+‐binding site. The simulations also confirm that if Ba2+ binds equally well to both open and closed states (that is, below the gate), there is no change in the rate or extent of block with Po (Fig. 3D, E). Experimentally, Ba2+ block was accelerated and decreased in its extent when the ATP concentration was increased (Fig. 2B). This effect can be simulated if Ba2+ binds below the gate and if, in the closed state, the off‐rate is faster and the on‐rate is increased by ATP (Fig. 3F). Thus, our results indicate that Ba2+ binds below the gate closed by ATP, and further suggest that Ba2+ binding is destabilized when the channel closes.
We tested the effect of increasing Po on Ba2+ block by using a mutant channel (Kir6.2‐C166S/SUR1) that exhibits a markedly enhanced Po (0.85; Trapp et al., 1998). If access to the Ba2+‐binding site is prevented when the channel is closed, the C166S mutation is predicted to accelerate the time course and increase the extent of Ba2+ block. However, no change in the rate of block by 3 μM Ba2+ was observed (τ = 22.6 ± 0.9 ms and τf = 23.5 ± 3.0 ms for mutant (n = 6) and wild‐type (n = 6) channels, respectively). Thus, these data provide additional support for the view that the gate lies above the Ba2+‐binding site.
Crystallographic studies of KcsA suggest that Ba2+ binds at the inner end of the selectivity filter, to the backbone of Val 76 and the side‐chain of Thr 75 (Zhou et al., 2001). The equivalent residues in Kir6.2 are Thr 130 and Ile 131 (Fig. 4A; Capener et al., 2000). Mutation of Thr 130 to valine in Kir6.2 did not result in functional channels, but mutation of Val 129 to threonine caused a marked decrease in Ba2+ affinity: the IC50 was 12 ± 0.2 μM (n = 5) compared with 0.8 ± 0.04 μM (n = 5) for the wild‐type channel (Fig. 4B). The maximum extent of block was 82 ± 1% (n = 5), indicating that Ba2+ might act as a partial blocker of V129T currents (Fig. 4B, C). These results confirm the Ba2+‐binding site of Kir6.2 lies within (or above) the selectivity filter, and indicate that the V129T mutation might modify the binding site allosterically. Studies on other Kir channels have also implicated this region in Ba2+ block (Lu et al., 2001; Alagem et al., 2001; Zhou et al., 1996).
Block of V129T channels by Ba2+ was well fitted by a single‐exponential curve (τ = 14.7 ± 2.0 ms at 55 μM free Ba2+; n = 5) that became faster with ATP (Fig. 4D–F). This allowed us to measure τ as a function of [Ba2+]i and to calculate the effective on‐rate and off‐rate constants of block (Fig. 4G). The on‐rate constant was little changed by the absence or presence of 15 μM ATP, being (9.3 ± 0.7) × 105 M−1 s−1 and (11.1 ± 0.1) × 105 M−1 s−1 (n = 5), respectively. In contrast, the off‐rate constant increased with ATP concentration, being 13 ± 5 s−1 in the absence of ATP and 22 ± 0.1 s−1 in the presence of 15 μM ATP (n = 5). These values predict a Kd of 14 μM Ba2+ in the absence of ATP, which is in good agreement with that found for steady‐state block (Fig. 4B). Thus, changes in off‐rate seem largely to account for the reduced extent of Ba2+ block found for V127T channels with increasing [ATP]i. This indicates that ATP modifies the Ba2+‐binding site, destabilizing cation binding.
Our results suggest that intracellular Ba2+ is still able to access its binding site when the KATP channel is closed by ATP, because the rate of block is not slowed when ATP is increased. Thus, the ATP‐dependent gate must be located above the Ba2+‐block site. Because Ba2+ binds at the inner end of the selectivity filter (probably near Thr 130), this puts the ligand‐sensitive gate within (or above) the selectivity filter. Because there is no obvious place higher up, we favour the idea that the gate lies within the selectivity filter (pore loop) itself, but our data are also consistent with the idea that the gate lies above the filter. Whichever is correct, the helix‐bundle crossing region at the internal mouth of the pore cannot serve as a gate for Ba2+. Because Ba2+ is almost the same size as K+ (crystal radii 2.7 and 2.66 Å, respectively), K+ must also be able to enter the pore when the channel is shut by ATP.
There is some evidence that the binding site for Ba2+ is modified when the channel is closed, because Ba2+ block is accelerated and decreased in amplitude when ATP is present. One possibility is that a conformational change consequent on channel closure accelerates the off‐rate of Ba2+ into the internal cavity. This might be more likely if the gate and the Ba2+‐binding site are located close together. Although our data place the gate for K+ ions at or above the selectivity filter, they do not exclude the possibility that large conformational changes take place at the helix‐bundle crossing region during gating of Kir6.2, nor that this region might serve as a gate for large molecules (such as thiol reagents).
ATP inhibits, and phosphatidylinositol (4,5)‐biphosphate (PtdInsP2) stimulates, KATP channel activity by altering the frequency of the long closed times (Trapp et al., 1998; Fan & Makielski, 1999). Thus, both agents probably modulate the same gate. Taken together, our data are consistent with the idea that this ligand‐operated gate is not at the helix‐bundle crossing region but resides within, or above, the selectivity filter (Fig. 1Ba). Thus, binding of ATP (or PtdInsP2) to the cytosolic domains of Kir6.2 must cause a conformational change that is propagated, through the transmembrane domains, up to the pore loop region. Gating of KATP channels, and possibly also other members of the inwardly rectifying K+ channel family, must therefore operate differently from that of KV channels (Del Camino & Yellen, 2001).
How might the selectivity filter serve as a gate for K+? One possibility is that the permeation path becomes physically occluded by one or more amino acids. An alternative idea is that electrostatic changes gate K+ flux through the pore, an effect that need not necessarily be associated with large changes in protein structure. A key question is whether the ligand‐dependent gating discussed here, and the intrinsic fast closings within bursts of openings (Proks et al., 2001), operate on the same or different gates.
Xenopus laevis oocytes were injected with ∼2 ng of SUR1 (GenBank accession number L40624) and ∼0.1 ng of wild‐type or mutant Kir6.2 (GenBank D50581) messenger RNAs (Trapp et al., 1998). Currents were measured 1–4 d later in excised inside‐out patches, at 18–24 °C, using an Axopatch 200B patch‐clamp amplifier. Macroscopic currents were filtered at 2 kHz and digitized at 5 kHz. Single‐channel currents were measured at +60 mV, digitized at 20 kHz and filtered at 5 kHz; their kinetics was analysed as described by Trapp et al. (1998). The external (pipette) and internal (bath) solutions contained (in mM): 107 KCl, 10 EGTA, 10 HEPES (adjusted to pH 7.2 with KOH). BaCl2 (in mM, given in parentheses) was added to the internal solution to give free concentrations (in μM) of 0.056 (0.01), 0.3 (0.05), 0.56 (0.1), 1.14 (0.2), 2.94 (0.5), 6.2 (1), 13.9 (2) and 55 (5). Free [Ba2+]i was calculated with the binding constants of Martell & Smith (1974). Concentrations in the text refer to free Ba2+ concentrations. Data are given as means ± s.e.m.
Ba2+ block of macroscopic currents was simulated with ModelMaker (Cherwell Scientific Publishing, Oxford). Rate constants for channel gating in the absence of Ba2+ or ATP were calculated from kinetic parameters measured in single‐channel studies, using a conventional model of KATP channel gating (Fig. 3B, D; Gillis et al., 1989). It was assumed that channel closure does not alter the mobility of ions in the parts of the pore that remain freely accessible to the bath solution.
We thank Chris Miller for critical advice, and the Beit Memorial Trust and Wellcome Trust for support. F.M.A. is the Royal Society GlaxoSmithKline Research Professor, P.P. is a Beit Memorial Fellow, and J.A. holds an MRC studentship.
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