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Correspondence to Christopher J. Lingle: clingle{at}morpheus.wustl.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zagotta et al., 1990) or portions of associated accessory ß subunits (Rettig et al., 1994; Heinemann et al., 1996; Xia et al., 1999, 2003). Inactivation provides a mechanism by which channel activity is self-limited under conditions that would otherwise lead to continuing ion conduction. A characteristic of simple models of pore occlusion is that inactivation is obligatorily coupled to opening of the channel's conduction pathway. However, there are indications of greater complexity in the inactivation of BK-type channels (Solaro et al., 1997; Lingle et al., 2001). In these cases, evidence suggests that the inactivation process is not well described by first-order association of an inactivating peptide with an open channel. Rather, the binding comprises multiple steps (Lingle et al., 2001), and the coupling of inactivation with opening may not be obligatory (Solaro et al., 1997). Here we investigate these properties further.
For BK-type potassium channels, expression of tetramers of the primary 
subunit produces functional channels that do not inactivate. Inactivation is conferred by the presence of either of two accessory subunits. In channels incorporating the ß2 subunit, inactivation requires an intact triplet of three hydrophobic amino acids, Phe-Ile-Trp (FIW), at the beginning of the ß2 NH2 terminus (Xia et al., 2003). In channels incorporating the ß3b subunit, inactivation also requires an intact NH2-terminal cytoplasmic tail in the accessory subunit, but the structural requirements have not yet been further defined (Xia et al., 2000).
Several lines of evidence suggest that inactivation of BK channels has significant differences with that of Shaker channels. Shaker channels transiently carry current during recovery (Demo and Yellen, 1991), and pore-blocking molecules compete for binding with the inactivation particle (Choi et al., 1991). In contrast, BK channels inactivated by the ß2 NH2 terminus can recover without passing through a conducting state, and no competition with pore blockers is observed (Solaro et al., 1997). Taken together, these observations suggest that the Shaker inactivation particle binds as a simple open channel blocker, while the BK inactivation domain may bind at a distinct, probably more superficial, site.
Another distinction between Shaker and BK inactivation is that inactivation mediated by the ß3b subunit does not occur as a simple, first-order binding of an inactivating particle into the pore. Specifically, properties of whole-cell currents require that channels containing the ß3b subunit possess a second, kinetically distinct conducting state that exists in rapid equilibrium with the inactivated conformation (Lingle et al., 2001). The simplest scheme accounting for these observations is shown below, with this additional short-lived conducting state designated O*:
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O*) followed by its further movement to occlude the mouth of the conduction pathway (O*I).
We have speculated that two-step inactivation may be a general feature of both ß2- and ß3-mediated inactivation, but that differences in the underlying rate constants preclude detection of this process in channels incorporating the ß2 subunit (Lingle et al., 2001). It has also been proposed based on structural considerations that Shaker and other KV channels inactivate through a similar multistep path (Zhou et al., 2001; Long et al., 2005). Here we further explore the generality of multistep inactivation. We demonstrate that the ß2 subunit also confers a second conducting state and a two-step inactivation process onto BK channels, and we use single channel recordings to visualize this short-lived state directly. A potentially physiologically significant aspect of these results is that channel opening is not absolutely required for the inactivation process to commence, and that channels can initially reach either the open or preinactivated states by kinetically distinct pathways.
Some of these data have been presented previously in abstract form (Benzinger et al., 2005).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2003).
Expression in Xenopus Oocytes
Expression of the clones used in this report has been previously described (Xia et al., 1999). 1 d after harvest, stage IV Xenopus oocytes were injected with 9–36 nl cRNA (10–20 ng/ml). To ensure saturation of expressed channels with ß subunits, ß subunit message was injected at a 2:1 excess by weight over subunit cRNA. Previous results have shown that a 1:1 ratio of ß2: subunit mRNA is sufficient to produce saturation of channels with ß subunits (Wang et al., 2002). For single-channel experiments we also used higher ratios (10:1) in some cases.
After injection, oocytes were maintained at 17°C in ND-96 medium (96 mM Na+, 2 mM K+, 1.8 mM Ca2+, 1 mM Mg2+, 103.6 mM Cl–, 5 mM HEPES, pH 7.5) with the following supplements: 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin. Oocytes were used 2–14 d after injection.
Electrophysiology
All currents were recorded in the inside-out patch clamp configuration (Hamill et al., 1981). Pipette resistance was typically 1–3 M
. The pipette solution comprised (in mM) 140 K-methanesulfonate, 20 KOH, 2 MgCl2, 10 HEPES, pH 7.2. High resistance seals were formed in a bath solution of ND-96. After development of a seal, the patch was pulled and immediately moved into a local perfusion stream containing (in mM) 140 K-methanesulfonate, 20 KOH, 10 HEPES, 5 HEDTA, pH 7.0. Ca-methanesulfonate was added to achieve a free Ca2+ concentration of 10 µM. Free [Ca2+] was verified through use of a Ca2+-sensitive microelectrode (Thermo Electron) and comparison to standard solutions (WPI) as previously described (Xia et al., 1999). For experiments involving treatment with cytoplasmic trypsin, trypsin (Sigma-Aldrich) at 1 mg/ml was added to the bath perfusate until modification of current morphology reached steady state, usually 3–5 min.
Currents were recorded using an Axopatch 200B amplifier and pClamp 9.2 software (Molecular Devices). Currents were filtered at 10 kHz through a 4-pole Bessel filter and digitized through a 16-bit DAC at 100 kHz. For macroscopic recordings, five instances of each voltage protocol were averaged and the results recorded. All experiments were performed at room temperature.
Data Analysis
Whole-cell recordings were analyzed using locally written software running under Scilab 3.0 (Scilab Consortium). Traces were usually analyzed without offline capacitance subtraction. However, for calculation of instantaneous current–voltage relations, the trace resulting from a step to a tail voltage of 0 mV was scaled appropriately and subtracted from the other sweeps in each recording.
Single-channel recordings were first baseline and capacitance corrected visually by subtracting a multicomponent decaying exponential. Corrected traces were interpolated 10-fold by a cubic spline procedure and idealized using a half-amplitude threshold criterion under pClamp 9.2, with idealizations verified visually. Approximately 1% of sweeps contained noticeable excursions to subconductance states; these sweeps were excluded from further analysis. Secondary analysis of the resulting events list was performed using locally written software. Simulated single-channel and macroscopic currents were generated from Markov schemes using the simulation functions of the QuB software package (State University of New York at Buffalo).
Rare reopening events in wild-type mSlo1+ß2 channels were analyzed using multichannel patches. In these cases, occasional overlapping reopenings were present. Because lengthy reopenings were rare, these overlaps almost always comprised a short, flickery event superimposed on a more lengthy reopening. Accordingly, the closure that terminated the excursion to the doubly open level was assigned to the opening event immediately preceding it.
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RESULTS |
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), BK channels incorporating the mSlo1 and ß3b subunits displayed rapid, time-dependent but incomplete inactivation, with significant steady-state current remaining even in the face of lengthy depolarizations (Fig. 1 A). The relative amplitudes of peak, steady-state, and tail currents changed as a function of potential (Fig. 1 B) (and see Uebele et al., 2000; Xia et al., 2000). The complex shape of the peak conductance–voltage relation has been observed previously, and is thought to be due to differing voltage dependences of the activation and inactivation processes (Xia et al., 2000). In contrast, channels incorporating the ß2 subunit inactivated somewhat more slowly, but virtually completely, with little steady-state or tail current (Fig. 1, C and D) (and see Xia et al., 1999).
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), and may therefore favor easier detection of a putative preinactivated state. Among these, we have focused on ß2-W4E because its properties seemed particularly amenable to elucidation of aspects of the proposed two-step inactivation mechanism.
Coexpression of the mutant ß2-W4E subunit with mSlo1 subunit produced channels with incomplete inactivation and large steady-state currents, as well as substantial tail currents upon repolarization (Fig. 1, E and F). Current decay was also somewhat faster in the mutant subunit. At +80 mV, decays were well described by a single exponential fit with 
= 35.5 ± 2.8 ms for ß2 (n = 11) and = 19.0 ± 3.5 ms for ß2-W4E (n = 10).
The behaviors of +ß2-W4E currents are qualitatively similar to those observed with the ß3b subunit, although the inactivation rate of ß3b substantially exceeded that of the wild-type and W4E ß2 subunits. Furthermore, for both ß3b and ß2-W4E, the tail upon repolarization had conductances that substantially exceeded the steady-state conductance measured at the conclusion of depolarization (in Fig. 1, B and F, compare triangles with circles).
Inactivation of +ß2-W4E Channels Exhibits Kinetics Inconsistent with Simple Open-channel Block
We investigated this rapid augmentation of tail current conductance further in Fig. 2. For many gating processes, it is assumed that the rate constants involved in channel gating are slow compared with the rate at which amplifiers can change command potential. Under this assumption, the number of open channels should remain constant over a voltage step, and the current present immediately before and after the step should scale as a simple function of the driving force. Wild-type mSlo1+ß2 channels display this approximately ohmic scaling, regardless of whether the duration of the depolarizing prepulse is short or long (Fig. 2 A). Similarly, mSlo1+ß2-W4E channels displayed approximately ohmic tail currents if the depolarizingprepulse preceding the voltage step was short and produced little inactivation (Fig. 2 B, black trace). However, when substantial inactivation was allowed to develop before returning to a negative command potential, the current present in the tail reflected an approximately twofold increase in conductance at the earliest measurable time point (Fig. 2 B, star). This augmentation must represent very rapid recovery of channels into a conducting state (summarized in Fig. 2 C).
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Both the nearly instantaneous recovery and the equally rapid redevelopment of inactivation occur at rates far in excess of inactivation and recovery rates assayed through traditional paired-pulse protocols in these channels (Xia et al., 2003). This nonohmic behavior is consistent with a very rapid, voltage-dependent equilibration of the inactivated state with a second conducting conformation that is distinct from the "classical" open state from which macroscopic inactivation proceeds.
At some voltages, tail currents of inactivated channels were also distinguished by a secondary unblocking component. After a lengthy depolarization that produced steady-state inactivation, recovery to moderately negative voltages (–130 to –20 mV) yielded tail currents with, in addition to the initial nonohmic enhancement, a secondary rising phase (Fig. 3 A). These tail currents were well fit by the sum of a rising and decaying exponential (Fig. 3 C). At a voltage of –80 mV, the potential used for the tail currents in Fig. 2, this rising phase had an apparent time constant of 5.1 ms (3.7–7.0 ms), which was too slow to account for the very rapid increase in tail current conductance noted above. Tail currents after a depolarization too short to produce substantial inactivation produced no such rising phase (Fig. 3 B). These currents were well fit with single exponentials, and they had time courses similar to the decaying phases of tail currents after long depolarizations.
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Properties of Instantaneous I/V Relations Require an Inactivation-dependent Second Open State
In any Markovian model with voltage-dependent parameters, the relative occupancy of the model's states after a voltage transition will change with time. In the case of Scheme 1, after the initial entry of channels into O, the ratio of occupancy of O and O* will change with time. Because O*, but not O, is assumed to be in a rapid, voltage-dependent equilibrium with the nonconducting I state, the instantaneous current–voltage relation is predicted to change as a function of the relative occupancies of O and O*. Therefore, the instantaneous I/V relation should change as a function of the duration of the depolarization preceding the voltage transition. In contrast, models incorporating only a single conducting state should have instantaneous I/V relations that do not vary with the duration of the prepulse.
Such a time dependence in the instantaneous I/V relation has already been observed in ß3b-mediated inactivation (Lingle et al., 2001). Although the instantaneous I/V curve for wild-type mSlo1+ß2 is relatively insensitive to prepulse duration (Fig. 4 A), channels incorporating ß2-W4E show a strong time dependence to their instantaneous I/V relations (Fig. 4 B). Application of trypsin to the cytoplasmic face of inactivating BK channels is known to eliminate inactivation (Solaro and Lingle, 1992). Accordingly, treatment of mSlo1+ß2-W4E patches with cytoplasmic trypsin produced noninactivating currents. Instantaneous I/V relationships for these patches were invariant with the duration of the depolarizing prepulse (Fig. 4 C). The occurrence of a second conducting state is therefore dependent on an intact inactivation pathway. The time dependence of the instantaneous I/V relation and its sensitivity to trypsin together require the presence of a second conducting state and are inconsistent with a model of inactivation based on simple open-channel blockade.
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O*I), and longer openings comprising more rare recovery into the longer-lived, classical conducting state (I[O*
O]I). Recordings from patches containing single mSlo1+ß2-W4E channels display this behavior (Fig. 5 A). Periods of numerous, poorly resolved flickery openings are interspersed with occasional longer excursions to the conducting state. Idealization of these data and construction of open-time histograms bear out this qualitative observation (Fig. 5 B). Open-time histograms have two very distinct primary components: a high-amplitude component comprising openings usually lasting <100 µs, and a smaller component of distinct, more lengthy openings typically lasting >1 ms.
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Occasionally, these patches also displayed lengthy quiescent states. (see, e.g., the last trace in Fig. 5 C.) These states were usually substantially longer than the latency before initial opening upon depolarization. It is therefore likely that this behavior represents an occasional, long-lived nonconducting state that is not described by any of the schemes considered here.
It is possible that the occurrence of an inactivation-dependent open state in mSlo1+ß2-W4E represents a phenomenon unique to this particular mutant and is not characteristic of the wild-type ß2 subunit. To answer this concern, we looked for the presence of similar short and long reopenings in wild-type mSlo1+ß2 channels. In macroscopic recordings, wild-type channels have little persistent current. Accordingly, recordings from patches containing single mSlo1+ß2 channels displayed reopenings very rarely. Occasionally, however, single brief, flickery reopenings were evident (Fig. 6 A, star). To view more of these events, we recorded prolonged depolarizations from patches containing 10–20 wild-type channels (Fig. 6 B). These recordings showed both frequent brief openings and uncommon longer openings. An open-time histogram constructed from idealized data revealed a bimodal distribution very similar to that associated with ß2-W4E (Fig. 6 C). The mean open time for the longer openings was 2.3 ± 0.5 ms (n = 3). Therefore, the wild-type ß2 subunit confers a preinactivated state onto BK channels in a manner similar to that of the W4E mutant.
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However, for mSlo1+ß2-W4E channels, the simple expectation of Scheme I does not hold. For example, the top sweep in Fig. 5 A shows an apparent initial opening that is very short lived. Even for Scheme I, very short initial openings would be occasionally expected as a matter of chance. To assess whether these short-lived initial openings occur more frequently than predicted, we compared an open-time histogram derived from all openings with one derived from only the initial opening in each depolarization (Fig. 7 A). Although the initial openings were significantly enriched in long-lasting events, the distribution of these openings remained significantly bimodal, indicating that, on average, + ß2-W4E channels open to the short-duration, preinactivated state 
39 ± 3% of the time (n = 4) patches. These channels are therefore readily able to pass directly to the preinactivated state without visiting the classical open conformation.
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lat = 2.1 ± 0.5 ms; for short openings, lat = 4.3 ± 1.1 ms (n = 4).
To illustrate this difference more directly, we constructed a scatter plot of the durations of the first latency and the initial opening for each sweep in a patch (Fig. 7 C). Openings were again separated as >200 µs (black) or 
200 µs (red). These distributions were log- transformed and averaged. The difference in latency for short events vs. long events when averaged in this manner, 3.9 vs. 2.3 ms in this example, was not large but was highly statistically significant (P < 0.0001, Student's t test). Data from four similar patches yielded an average latency preceding short events of 3.4 ms (2.9–4.0, mean ± SEM), and for long events of 1.9 ms (1.5–2.4, mean ± SEM). We therefore conclude that, for +ß2-W4E channels, the path toward inactivation cannot be represented by a purely linear sequential model, and that channels may pass through different pathways before initially opening to the O or O* states.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both macroscopic and single-channel data provide evidence for the existence of this state. Channels including mutant ß2 subunits display apparent nonohmic increases in tail current, consistent with redistribution between I and O*, and instantaneous current–voltage relationships differ with the relative proportions of channels in the O and O* states. Both brief reopenings (to O*) and lengthy reopenings (to O* and O) are visible in single-channel recordings from both wild-type and mutant channels. Finally, enzymatic treatment to remove inactivation removes the preinactivated state.
Structural Correlates of the Preinactivated State
The simplest structural explanation consistent with these observations is that the ß2 NH2 terminus produces inactivation through a multistep blocking process. In this model, the preinactivating transition corresponds to the binding of the NH2 terminus in close association with the pore, while full inactivation occurs when the NH2 terminus moves further and occludes it. The location of the initial binding site remains unknown. However, the short duration of openings to the preinactivated state suggests that the site must be close to the final binding site for full inactivation.
Is this model consistent with the structural features of the BK channel cytoplasmic face? Recent structural work with KV channels has suggested that the Shaker inactivation domains reach the ion-conducting pore through lateral "side portals" in the cytoplasmic portion of the channel (Gulbis et al., 2000; Long et al., 2005). BK channels also possess large cytoplasmic domains tethered to the cytoplasmic end of the S6 helix. It therefore seems likely that access of the BK inactivation particle to the conduction pathway occurs through side portals, as well. Mutagenesis experiments with the BK ß2 NH2 terminus have shown that many different amino acid sequences between the hydrophobic triplet and the transmembrane region will support inactivation (Xia et al., 2003). In addition, an NMR structure for the NH2 terminus showed considerable flexibility over much of this region (Bentrop et al., 2001). These facts together suggest that the NH2-terminal inactivation domain may be sufficiently pliant to make its way through a postulated side portal.
The limited length of the BK inactivation domain also suggests that it may access the conduction pathway through a side portal. Mutagenesis studies in ß2 have shown that a linker region of only 12 amino acids between the membrane and the hydrophobic triplet is sufficient to support functional inactivation (Xia et al., 2003). In a maximally extended conformation, this minimally acceptable 15–amino acid linker can extend 57 Å (Creighton, 1993). For comparison, the cytoplasmic "T1" domain of KV1.2 is 40 Å in height (Long et al., 2005), and BK subunits have a cytoplasmic domain that is likely to be considerably larger than that of Shaker-type channels (Stuhmer et al., 1989; Butler et al., 1993). Given such a large cytoplasmic region, access to the conduction pathway through a side portal seems very likely.
If the inactivation domain enters the channel in this manner, ample opportunity exists for interaction between the inactivation domain and the subunit before the pore is occluded. Preinactivation may represent the interaction of the ß2 NH2-terminal residues with the lining of the side portal, followed by full inactivation as the NH2 terminus advances into the pore. Alternately, preinactivation may involve the interaction of the hydrophobic triplet within the channel's central axis, but at a more superficial, nonoccluding position than that associated with inactivation. In either case, excursions to the preinactivated state are very short, while the macroscopic rate of channel inactivation is considerably slower. These facts imply that entry from the open state into the preinactivated state is the rate-limiting step in channel inactivation. The ß2 NH2 terminus in its preinactivated position therefore must be very close to its inactivating, occlusive conformation. Given the side portal structure of KV channels, it is perhaps more surprising that Shaker inactivation appears to occur as a first-order association process. Further work with Shaker may reveal intermediate steps in its inactivation pathway, as well (Zhou et al., 2001).
The proposed movement of the ß2 inactivation particle through a side portal raises additional questions related to the stoichiometry of channel inactivation. Previous work using low levels of ß2 mRNA has shown that the presence of a single ß2 subunit in the channel complex is sufficient to permit inactivation, and that additional ß2 subunits increase the rate of inactivation in a dose-dependent manner (Wang et al., 2002). The presence of four side portals and four ß2 subunits admits the possibility that multiple ß2 NH2 termini can exist in a preinactivated conformation, contained within multiple side portals, simultaneously. Alternately, if the preinactivated conformation involves a degree of impingement into the channel's central axis, it may be that preinactivation is competitive, and that multiple NH2 termini cannot concurrently occupy this state. We believe that these alternatives are potentially experimentally distinguishable.
Ability of BK Channels to Inactivate without First Opening
Our results indicate that fully closed channels bearing the ß2-W4E subunit can occasionally open directly into the preinactivated state without ever passing through a long-lived open conformation. This observation is incompatible with the strict linear sequential model given in Scheme 1. Previous studies have shown that BK channels need not conduct current during recovery from inactivation, implying that closure of a channel's activation gate is not impeded by the presence of a docked inactivation particle (at least during recovery) (Solaro et al., 1997). This result suggests that even the final binding site of the inactivation particle within the pore is more superficial than that usually envisioned for Shaker channels. Therefore, the simplest model explaining these data allows for mSlo1+ß2-W4E channels to reach (at least) the preinactivated state before channel opening:
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In this model, movement of the inactivation particle into the preinactivated position can precede opening of the activation gate (CC*O*), yielding an initial brief opening. Because wild-type mSlo1+ß2 channels show very few direct openings to the short-lived O* state, it is natural to ask whether Scheme 3 only applies to the mutated variant that we have studied. Prior studies on native BK channels in adrenal chromaffin cells, which most likely include the ß2 subunit, have shown that, under conditions of weak activation, these channels can inactivate without ever opening (Ding and Lingle, 2002). Transition through the classical open state was therefore not an absolute consequence of depolarization in these channels, either.
Is it then possible to account for the empiric differences in single-channel behavior between wild-type and mSlo1+ß2-W4E channels upon initial activation within the context of Scheme 3? We are aware of two possibilities. First, wild-type channels may have a slower rate of preinactivation (CC*) with respect to activation (CO) than is the case in mSlo1+ß2-W4E channels, thereby favoring full openings upon depolarization. In support of this idea, we observed that macroscopic current decay after depolarization was approximately twofold slower at +80 mV for channels including wild-type ß2 than for ß2-W4E. This result suggests that the preinactivating process is slower in wild-type channels than in channels containing ß2-W4E.
Second, it is possible that, despite our hyperpolarizing prepulses, some mSlo1+ß2-W4E channels remain inactivated at the beginning of our depolarizations. In contrast, wild-type channels might be fully available by the onset of depolarization. Although we cannot exclude this possibility, we do not consider it likely. In 10 µM Ca2+, BK channels incorporating wild-type ß2 are virtually fully available at potentials more negative than –120 mV (Ding and Lingle, 2002). Our prepulse occurred at –160 mV, providing a considerable margin of safety. Furthermore, despite the steady-state conductance properties of mSlo1+ß2-W4E, very little current is carried by these channels at the end of the hyperpolarizing prepulse, suggesting that few, if any, channels remain open or inactivated at this point (see Fig. 1 C).
We therefore prefer the view that the observed difference in the tendency of wild-type and mSlo1+ß2-W4E channels to open directly to the preinactivated state is explicable within Scheme 3. It likely represents a difference in the relative rates of the opening and preinactivating transitions, a difference that is consistent with other known macroscopic data.
To assess the ability of Scheme 3 to explain the observed properties of mSlo1+ß2-W4E current, we constructed a quantitative model and assessed the resulting simulated events. The process of BK channel activation encapsulates the movement of up to four voltage sensors and the binding of up to four Ca2+ ions and is therefore represented by a Markov scheme comprising many dozens of states (Horrigan and Aldrich, 2002). The rate constants associated with activation are not well constrained by single-channel data obtained at single values of voltage and [Ca2+]. For the present purpose, however, at least one additional resting state (denoted CR) was required to adequately model the latency preceding initial channel opening. The resulting six-state model is shown in Fig. 8 A.
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This model also predicted the ability of channels to open initially into the preinactivated O* state. However, channels followed this path with a rate much lower than the 39% observed experimentally. This discrepancy arises from the interaction of two of the observed kinetic parameters. A higher fraction of channels initially opening into O* predicts a higher rate of closed state preinactivation (CC*), while a longer duration of long-lived bursts predicts a lower rate of open state preinactivation (OO*). Permitting the presence of (negative) allosteric coupling between channel opening and preinactivation allows for a higher rate of openings into the preinactivated state without limiting the burst length of longer openings. The extent and physical basis of this allostery may prove a fruitful area for further study.
The presence of short-lived initial openings implies that channels can reach the preinactivated state without passing through the classical open conformation. This process might arise either from preinactivation of a fully closed channel or from an initial voltage-dependent conformational change that precedes channel opening and favors preinactivation. Previous studies have shown substantial steady-state levels of inactivation of BK channels incorporating the ß2 subunit at potentials for which activation was minimal (Ding and Lingle, 2002). However, the presence of a closed, fully inactivated state is not necessary to explain the current and previous results. Macroscopic rates of inactivation display only a very modest voltage dependence, despite the obvious voltage dependence of steady-state availability curves (Ding and Lingle, 2002). Further, charged residues are not required on the ß2 NH2 terminus to produce inactivation (Xia et al., 2003). The apparent development of steady-state inactivation in the absence of conductance could therefore reflect channel entry into the closed, preinactivated conformation (C*), followed by full inactivation almost immediately upon channel opening during the test pulse.
An important implication of this idea is that a structural transition associated with activation but preceding channel opening is required to permit movement of the NH2 terminus into its full inactivating position. One possible candidate for this transition is the movement of the S4 voltage sensor. Current evidence suggests that substantial gating charge movement occurs very rapidly upon depolarization and significantly before the onset of channel opening (Horrigan and Aldrich, 1999). Therefore, S4 movement is well poised to act as a permissive switch for channel inactivation by the ß2 NH2 terminus.
A cartoon encapsulating these ideas is shown in Fig. 8 D. In this scheme, a conformational change associated with movement of the voltage sensor is permissive for preinactivation and precedes channel opening. Preinactivation is represented by movement of the ß2 NH2 terminus into a side portal, followed by binding at a position close to the ion conduction pathway. Further movement into the pore then corresponds to complete channel inactivation. By permitting inactivation without first passing through the open state, this multistep inactivation process may help regulate levels of BK current across a range of voltages in tissues in which inactivating BK accessory subunits are found.
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ACKNOWLEDGMENTS |
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This work was supported by grant DK-46564 from the National Institutes of Health (C.J. Lingle) and by the Foundation for Anesthesia Education and Research (G.R. Benzinger).
Lawrence G. Palmer served as editor.
Submitted: 6 October 2005
Accepted: 22 December 2005
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REFERENCES |
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and auxiliary ß subunits on functional properties of large-conductance Ca2+-activated K+ channels. J. Neurosci. 22:1550–1561.
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), steady-state (
), and tail (
) currents were measured. Peak currents 
2 ms). For openings >0.2 ms (black), 
