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Correspondence to Robert Brenner: brennerr{at}uthscsa.edu
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subunits and a family of tissue-specific accessory ß subunits. The smooth muscle–specific ß1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of ß1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of ß1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca2+ binding) due to the very low open probability in the presence of ß1. In this study, we used a mutation of the subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the ß1 subunit. Effects of ß1 on steady-state open probabilities of both wild-type and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse ß1 has two major effects on channel's gating energetics. ß1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca2+. Further, PO measurements at limiting slope allow us to infer that ß1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca2+ concentrations. Using the F315Y subunit with deletion mutants of ß1, we also demonstrate that the small N- and C-terminal intracellular domains of ß1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that ß1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.
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INTRODUCTION |
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250 pS) and are therefore very effective in hyperpolarizing the membrane. The coincident activation of BK channels by Ca2+ and voltage makes these channels uniquely tailored to regulate voltage-dependent Ca2+ channels in a number of cell types (Kaczorowski et al., 1996
; Gribkoff et al., 1997; Calderone, 2002). BK channels in smooth muscle use the accessory ß1 subunit to promote channel opening (Knaus et al., 1994; Tanaka et al., 1997). Previously the important role of the ß subunit has been demonstrated by targeted gene knockout of the ß1 locus in mice. Knockout mice have BK channels with reduced openings, increased vascular tone, and hypertension (Brenner et al., 2000b; Pluger et al., 2000).
BK channel open probability is dependent on its intrinsic closed to open equilibrium that is described by the equilibrium constant L (Horrigan and Aldrich, 2002). This is the inherent PO of the channel without influence of other gating mechanisms. BK channel gating is also allosterically coupled to voltage sensor activation and Ca2+ binding (Horrigan and Aldrich, 2002). A prominent effect of ß1 subunits is an increase in BK channel openings. However it is not well established how, and to what degree ß1 subunit effects on L, voltage sensor activation, or Ca2+ binding contribute to enhanced PO.
Historically, because ß1 causes a negative voltage shift of the conductance–voltage (G-V) relationship, in a manner similar to increased Ca2+, the effects of the ß1 subunit was first described as an "increase in apparent Ca2+ sensitivity" (McManus et al., 1995; Dworetzky et al., 1996; Meera et al., 1996). Later, it was found that this effect may not be due exclusively to changes in Ca2+ binding equilibrium (Cox and Aldrich, 2000; Nimigean and Magleby, 2000; Bao and Cox, 2005; Orio and Latorre, 2005). Using gating current measurements, Bao and Cox clearly demonstrated that the bovine ß1 subunit shifts voltage sensor activation to more negative membrane potentials, and this may account for ß1 enhanced openings (Bao and Cox, 2005). Orio and Latorre (2005) also suggested that human ß1 shifts open channel voltage sensor activation to more negative membrane potentials. Effects of ß1 on channel's intrinsic gating are less clear. Whereas Orio and Latorre proposed that human ß1 reduces channel's intrinsic equilibrium (L) for opening, Bao and Cox suggested otherwise for the bovine ß1.
Based on HA model for BK channel gating (Horrigan and Aldrich, 2002), it is advantageous to directly compare and +ß1 under conditions that isolate the influence of intrinsic gating. This is accomplished by measuring ionic current at 0 Ca2+ (to exclude effects on Ca2+ binding) and very negative membrane potentials (the limiting slope, to exclude effects on voltage sensor activation). Measurement at higher voltages can then indicate the contribution of voltage sensor activation. This approach has proven useful for evaluating BK channel subunits alone (Horrigan and Aldrich, 2002; Ma et al., 2006). However, under such conditions, ß1 channel openings fall below detection levels and this approach has not been feasible (Bao and Cox, 2005; Orio and Latorre, 2005).
Here, we have used a previously described subunit mutation (F380Y in human cDNA) (Lippiat et al., 2000) that increases channel openings to investigate ß1 subunit effects on channel gating. This allows, for the first time, measurement of +ß1 PO in the absence of Ca2+ and voltage sensor activation. Analysis of PO-V relationships using the dual allosteric HA gating model revealed that the ß1 subunit confers two opposing effects on channel openings; both a negative voltage shift for voltage sensor activation (VhO) that contributes to increased channel openings seen in micromolar Ca2+, and a reduced closed to open equilibrium (L0) that contributes to reduced channel openings seen in submicromolar Ca2+. Further, deletion analysis demonstrates that interactions at the small intracellular domains mediate intrinsic and voltage-dependent gating effects of ß1.
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MATERIALS AND METHODS |
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) and mouse cDNAs (GenBank/EMBL/DDBJ accession no. MMU09383) were cotransfected into HEK293 cells. The F380Y mutation, originally described in the human cDNA (Lippiat et al., 2000), was introduced in the mouse subunit cDNA (site is F315Y in mouse) using the Stratagene Quick-Change Mutagenesis kit.
Mouse ß1 mutants were generated by PCR amplification of the ß1 cDNAs with amplification primers that delete the N-terminal residues KKLVMAQKRGE (residues 3–13) and C-terminal residues NRSLSILAAQK (residues 181–191) for ß1
N11 and ß1C11, respectively. The double mutant, ß1N10C11 differs in that the E13 residue was not deleted. Using a C-terminal epitope-tagged ß1N11C11, immunostaining showed expression. However, electrophysiology recordings showed no evidence of functional interactions with BK subunits using stimulus protocols and a broad range of calcium as in Fig. 1.
Mutant and wild-type mouse ß1 subunits were subcloned in the mammalian expression vector pIRES2-EGFP (CLONTECH Laboratories, Inc.), which fluorescently labels cells with channel expression. The mouse subunit was cotransfected at a ratio of 1:10 to ß1 to ensure saturation of BK channels with ß1 subunits.
Macropatch recordings were made using the excised inside-out patch clamp configuration. To limit series resistance errors, currents 5 nA or less were used for steady-state G-V and analysis of channel kinetics. Experiments were performed at 22°C. Data were sampled at 10–30-µs intervals and low-pass filtered at 8.4 kHz using the HEKA EPC8 four-pole bessel filter. Data were analyzed without further filtering. Leak currents were subtracted after the test pulse using P/5 negative pulses from a holding potential of –120 mV. For BK/+ß1, leak subtraction was not performed at 18.5 and 100 µM Ca2+. Patch pipettes (borosilicate glass VWR micropipettes) were coated with Sticky Wax (Kerr Corp.) and fire polished to 1.5–3 M
resistance.
The external recording solution (electrode solution) was composed of 20 mM HEPES, 140 mM KMeSO3, 2 mM KCl, 2 mM MgCl2, pH 7.2. Internal solutions were composed of a pH 7.2 solution of 20 mM HEPES, 140 mM KMeSO3, 2 mM KCl, and buffered with 5 mM HEDTA and CaCl2 to the appropriate concentrations to give 1.7, 7, and 18.5 µM buffered Ca2+ solutions. Higher Ca2+ solutions were buffered with 5 mM NTA. Low Ca2+ solutions (0.3 µM and 0 Ca2+) were buffered with 5 mM EGTA, and Ba2+ was chelated with 40 µM (+)-18-crown-6-tetracarboxylic acid (Cox et al., 1997b). Free [Ca2+] of buffered solutions were measured using an Orion calcium-sensitive electrode (Orion Research, Inc.).
Analysis of Macroscopic Currents
Conductance–voltage (G-V) relationships were obtained using a test pulse to positive potentials followed by a step to a negative voltage (–80 at low Ca2+, –120 at high Ca2+), and then measuring instantaneous tail current 200 µs after the test pulse. In experiments where Gmax were not reached, including BK/+ß1 and BK/+ß1N11 at 0.005 and 0.3 µM [Ca2+], BK/+ß1C11 and BK/+ß1N10C11 at 0.005 µM [Ca2+], Gmax values at higher [Ca2+] from the same patch were used. G/Gmax-V data were fitted with the Boltzmann function: G = Gmax{1/[1 + e–(V – V1/2)ZF/RT]}, where V is the test potential, V1/2 is the membrane potential at half-maximal conductance, z is the effective gating charge, and F, R, and T are constants.
Single Channel Analysis
Single channel opening events were obtained from patches containing one to hundreds of channels. Recordings are of 20 s to hundreds of seconds duration. Analysis was performed using TAC and TACFIT programs (Bruxton Corporations). NPO was determined using either all-point amplitude histogram or by event detection using a 50% amplitude criteria. The probability (Pk) of occupying each open level (k) give rise to NPO:
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PO was then determined by normalizing NPO values by channel number (N). N was obtained from the instantaneous tail current amplitude during maximal opening at saturating [Ca2+], divided by the unitary conductance for each channel at the tail voltage. Combined single channel and macroscopic steady-state data in 0 Ca2+ in the presence of F315Y mutation were fit with the dual allosteric model assuming voltage-dependent transitions only (Horrigan and Aldrich, 2002). Details for fitting parameters are included in figure legends.
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RESULTS |
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subunit alone (BK/) or with saturating mß1 expression (BK/+ß1) were transiently expressed in HEK293 cells, and macroscopic BK currents were recorded in the inside-out patch clamp configuration. BK currents were evoked by step depolarization at controlled intracellular Ca2+ (Fig. 1, A and B, left panels) to obtain normalized steady-state tail conductance versus voltage (G-V) relationships (Fig. 1, A and B, right panels). Averaged V1/2-Ca2+ and Q-Ca2+ relationships obtained from Boltzmann fits of the G-V relationship (Fig. 1, C and D) show that mß1 subunit alters V1/2 and Q in a Ca2+-dependent fashion. In the presence of mß1, there is a steeper V1/2-Ca2+ relationship (Fig. 1 C) that indicates an increase in apparent Ca2+ sensitivity. Below 1.7 µM Ca2+, mß1 subunit shifts the G-V relationships to positive potentials. This is most dramatic at nominal 0 Ca2+, where G/Gmax for BK/+ß1 channels only reaches 0.23 at 300 mV. Extrapolation of the V1/2 from the Boltzmann fit predicts that mß1 confers an 150-mV positive shift in V1/2. Above 1.7 µM Ca2+, however, mß1 causes a negative shift in the V1/2 (–50 mV shift at 100 µM Ca2+). In addition, the mß1 subunit reduces the apparent equivalent gating charge (Q) at low Ca2+ (Fig. 1 D).
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; Horrigan and Aldrich, 2002). In this model, channel opening is governed by three equilibrium constants, L (closed-to-open transition), J (voltage sensor activation), K (Ca2+ binding), and D, C, and E, the allosteric couplings between L and J, L and K, and J and K, respectively. Open probability is described by Eq. 1, referring to the HA model (Horrigan and Aldrich, 2002):
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In the absence of Ca2+, the occupied states are reduced to 10 (Fig. 2 A, left),
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In the absence of Ca2+ and at extremely negative membrane potentials (limiting slope), virtually all voltage sensors reside in the resting state and the occupied states are further reduced to 2 (C0 and O0) (Fig. 2 A, dashed box).
Because J is small (J << 1/D), Eq. 2 reduces to
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In this equation, zL is the voltage dependence of the closed-to-open transition and L0 is channel's closed-to-open equilibrium in the absence of Ca2+ and voltage sensor activation at 0 mV (Fig. 2 B). Therefore a direct approach to evaluate effects of ß1 on channel's intrinsic closed to open equilibrium is to compare logPO-V at 0 Ca2+ and limiting slope. As predicted by Eq. 4, the position of the limiting slope of logPO along the Y axis is determined entirely by L and zL (Fig. 2 C).
Another advantage of PO measurement near the limiting slope is that it allows one to infer effects on open-channel voltage sensor activation (VhO, see Table I for definitions). As shown in Fig. 2 D, the HA model predicts that membrane potentials where PO transitions from weakly voltage dependent to "steep" voltage dependence is critically dependent on VhO and relatively unaffected by other voltage-dependent parameters, including closed channel voltage sensor activation (VhC) or the charge associated with voltage sensor activation (zJ).
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20-fold) and gap duration (2–3-fold) for a net sevenfold increase in PO (Nimigean and Magleby, 2000). At a similar voltage (+40 mV), we also found that mß1 increases both mean burst duration (sixfold) and mean gap duration (fourfold) (Fig. 3, B and C). This resulted in a PO that is similar for /mß1 vs. channels (6.3 e–5 ± 2 e–5 /mß1 vs. 6.3 e–5 ± 0.8 e–5 ; Fig. 3, D and E). However, at negative voltages, although the fold change in mean burst duration is somewhat reduced (3.6-fold at –60 mV), mß1 causes much longer gaps between open events (3 e4 ± 0.9 e4 s BK/+ß1 vs. 4.4 e2 ± 9 e1 s BK/, –60 mV; see Fig. 3 C). The larger increase in gap duration (70-fold) at negative voltages likely underlies a 15-fold reduction in PO of BK/+ß1 channels over BK/ channels (7.4 e–8 ± 5 e–8 vs. 1.1 e–6 ± 2.7 e–7, respectively).
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(Fig. 3 D) and BK/+ß1 (Fig. 3 E) indicates that there are two differences between these channels' steady-state properties. First, whereas the logPO-V curve of BK/ displays a clear transition in voltage dependence (approached limiting slope at approximately +40 mV), logPO-V curve of BK/+ß1 does not. Based on the HA model, this result suggests that mß1 shifts VhO to more hyperpolarized membrane potentials (Fig. 2 D). In addition, logPO at negative voltages for BK/ is substantially greater than BK/+ß1, indicating a decreased closed to open equilibrium (L0) in the presence of mß1. To quantify mß1-mediated changes in L0, VhO, and VhC, data in Fig. 3 (D and E) were fitted using Eq. 2, where zL and zJ were held at 0.30 e0 and 0.58 e0, respectively, based on previous estimates (Horrigan and Aldrich, 2002; Bao and Cox, 2005; Wang et al., 2006). For BK/, estimated L0, VhO, and VhC were 1 e–6, +46 mV, and +202 mV, respectively (Table II).
These values are reasonably close to previous estimates (Horrigan and Aldrich, 2002; Bao and Cox, 2005; Wang et al., 2006). For BK/+ß1, because PO drops so dramatically (PO < 10–8), it was not technically feasible to obtain PO at the limiting slope. Therefore, existing data only provides estimates for the upper and lower limits for L0 (between 1 e–10 and 1 e–8) and VhO (between –70 and –20 mV), whereas VhC is estimated to be +130 mV. We also attempted to improve the fitting for BK/+ß1 by setting equivalent gating charge for voltage sensor activation (zJ) to a lower value. Previously, others had found that some ß1 effects on BK channels could be explained by reducing zJ to 0.37 e0, (Orio and Latorre, 2005). However, we found that holding zJ to 0.37 e0 produces a poor fit of BK/+ß1 data (Fig. 3 F), which suggests that mß1 does not lower zJ.
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; Fig. 3 E and Table II). In both cases, ß1 shifts VhC to hyperpolarized membrane potentials by 70 mV. Gating current measurements found that bß1 shifts VhO by –60 mV (Bao and Cox, 2005) and our fits estimate that mß1 causes a VhO shift between –20 and –70 mV. Effects of mß1 on L0, however, differ from that proposed for bß1. Whereas a >100-fold decrease in L0 was estimated for mß1, it was proposed that bß1 slightly increases L0. Because PO measurement also did not reach the limiting slope in the study performed by Bao and Cox (2005), it is not clear whether bß1 indeed increases L0. Effects of hß1 on channel gating was also investigated using ionic currents in the context of the HA model (Orio and Latorre, 2005). The authors proposed that hß1 significantly decreases L0, VhO, and zJ, with little effects on VhC.
F315Y Mutation Dramatically Increases Channel's Closed-to-Open Transition
The above analysis indicates that mß1 subunits have effects on BK channels that should be apparent at limiting slope. However, the greatly reduced PO combined with the negative voltage shift of VhO make PO measurements at limiting slope not feasible. Previously it had been shown that F380Y, a point mutation in the S6 transmembrane domain of hslo, significantly increases PO even at 0 Ca2+ (Lippiat et al., 2000). The F380 residue lies in a position within the C-terminal domain of S6 that may serve as the gate for Kv channels (Swartz, 2005). An mslo equivalent of the F380Y mutation was generated (F315Y in mouse) and characterized at 0 Ca2+ using macroscopic and single channel recordings (Fig. 4 A).
Similar to previous findings, F315Y shows extremely long open dwell times, (Fig. 4 A, left panel vs. Fig. 4 B). For example, open burst durations are 11 ± 2 ms for F315Y vs. 0.36 ± 0.02 ms for WT at –60 mV. Similar to previous results, F315Y produces a dramatic leftward shift in the G-V relationship and a decrease in the apparent voltage dependence (Fig. 4 C) (Lippiat et al., 2000). Fitting individual logPO data at limiting slope using Eq. 4 estimated a slight reduction in zL (0.30 wild type , 0.26 ± 0.04 e0 for F315Y, n = 6). Fitting both logPO and PO data using Eq. 2, gating parameters L0, zJ, VhC, and VhO are estimated to be 9 e–2, 0.58 e0, +92 mV, and +35 mV, respectively (Fig. 4 F and Table II). The large decrease in VhC and little change in VhO decreases D from 35.2 to 3.7, which explains the shallower G-V slope (apparent voltage dependence) for F315Y. To rule out the possibility that the reduced G-V slope can be explained by a reduction in zJ alone, we also fit the F315Y data by holding VhC and VhO at wild-type values, and zL at 0.26 e0 (estimates from limiting slope measurements) (Fig. 4 G). This yielded a poorer fit. In summary, these results indicate that the F315Y has two effects. These are a negative voltage shift of VhC and a greater than 104 increase in L0 relative to wild-type subunits. We next used the large increase in L0 in F315Y to investigate mechanisms underlying BK channel modulation by the ß subunits at limiting slope.
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+ß1, BK/F315Y+ß1 maximal PO (1) (+20 to +40 mV) and maximal conductance at 0 Ca2+ can be easily observed (Fig. 5, B and C). Averaged G-V relationships (Fig. 5 C) suggest that mß1 shifts the V1/2 to more depolarized membrane potentials, with a slight increase in the slope of the G-V relation. LogPO-V curves of individual patches were fitted using Eq. 4. Similar to wild-type BK channels, mß1 does not significantly alter zL of F315Y (Fig. 5 E and Table II; BK/F315Y 0.26 ± 0.04 e0, BK/F315Y+ß1 0.27 ± 0.04 e0, P = 0.64). We estimated VhC, VhO, and L0 by fitting both PO-V and logPO-V data using Eq. 2. VhC and VhO were estimated to be 72 and –26 mV, respectively (Fig. 5 F and Table II). This is a –20-mV shift of VhC and –61-mV shift of VhO over BK/F315Y channels. Effects on voltage sensor activation estimated by our fits are qualitatively similar to bß1 measured directly using gating current measurements (Bao and Cox, 2005). However, although shifts of VhO are similar, the –20-mV shift of VhC in the F315Y background is smaller than the –71-mV shift measured by Bao and Cox (2005). Consistent with mß1 effects on WT subunit (Fig. 3 E), mß1 also caused a dramatic (50-fold) reduction of intrinsic gating in the BK/F315Y subunit (Fig. 5 F). L0 for BK/F315Y is 9 e–2 versus 1.8 e–3 for BK/F315Y+ß1. In summary, the F315Y mutation allowed us to measure effects on intrinsic gating by mß1 despite the dramatic reduction in PO. Further, extending PO measurement to the limiting slope provides an assay to measure effects of voltage sensor activation on PO and thereby constrain estimates of VhO using the HA model.
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subunits, mß1 causes a positive G-V shift in low Ca2+ and a negative G-V shift in high Ca2+, with a crossover of the V1/2 around 1.7 µM Ca2+ (Fig. 1 C). This creates a steeper V1/2-Ca2+ relationship. How does ß1 modulation of L0 and VhO contribute to these properties? We simulated wild-type subunit PO (HA model, Eq. 1) across a range of Ca2+ by varying L0, VhO, or both VhO and VhC, either individually or in combination, to understand their effect on the V1/2-Ca2+ and Q-Ca2+ relations (Fig. 6).
As shown in Fig. 6 A, reducing L0 by mß1 causes a positive shift of the G-V to a lesser extent at high Ca2+ than at low Ca2+, causing the V1/2-Ca2+ relationship to be more steep. In addition, reducing L0 also reduces Q at low Ca2+. This is because the decrease of L0 causes significant channel openings to occur at much more positive potentials than VhC where voltage-dependent gating rely on the weak voltage dependence of the closed-to-open transition, zL (Wang et al., 2006). Thus, the reduced intrinsic gating creates a double hit to inhibit channel openings: a greater energetic barrier due to L0 and a much weaker voltage dependence (Q) as significant channel openings occur more much positive than VhC (Fig. 6 A, right). Therefore the V1/2 is shifted to far positive values. With the contribution of higher Ca2+ (>1.6 µM), channel openings fall within the range of voltage sensor activation (between VhO and VhC) and the effect of decreased L0 on V1/2 is greatly reduced and fairly uniform across 1.7–100 µM Ca2+. We can see that the HA model predicts that shifting VhC to more positive potentials (Fig. 6 B, e.g., +400 mV) places channel openings within the effective range of voltage sensor activation despite the decrease of L0. In that case, effect of L0 on V1/2 is uniform across both low and high Ca2+ concentrations. Thus, the HA model predicts that mß1 effects on L0 contribute to a much larger positive shift of the V1/2 and reduced voltage dependence (Q) at low Ca2+ than high calcium, which would steepen the V1/2-[Ca2+] relations.
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Intracellular Domains of ß1 Are Required for ß1-mediated Modulation of Voltage-dependent Gating
The ß1 subunit is composed of a large extracellular domain and small N- (15 amino acids) and C-terminal (12 amino acids) domains. Given that the intracellular domains of the subunit are required for ß1 subunit–mediated G-V shift (Qian et al., 2002), the ß1 intracellular domains were deleted to evaluate their role in modulating gating. 11 amino acids that follow the N-terminal initiating methionine and glycine were deleted in ß1N11 (Fig. 7 A).
In addition, the C-terminal 11 residues were deleted in ß1C11 (Fig. 7 E). Effects of ß1N11 and ß1C11 on steady-state gating of wild-type subunit were examined over a wide range of Ca2+ (Fig. 7, B, C, F, and G). These data are summarized in V1/2-Ca2+ and Q-Ca2+ plots (Fig. 7, D and H). Surprisingly, deletion of either intracellular domain has similar effects on the G-V relationship. Both mutants eliminate the negative voltage shift of the G-V relationship in high Ca2+, but maintain the positive G-V shift to varying extents in low Ca2+ (Fig. 7, D and H).
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N11 and ß1C11 were coexpressed with BK/F315Y to examine whether the mutations affect ß1's ability to reduce L0 and VhO. Macroscopic and single channel recordings (Fig. 8) were used to obtain the PO-V relationship.
Fitting the logPO-V relationship (Fig. 8, B and E) at limiting slope using Eq. 4 estimated that zL for both ß1N11 (0.24 ± 0.05 e0) and ß1C11 (0.24 ± 0.05 e0) is not significantly different from wild-type ß1 (0.27 ± 0.04 e0, P = 0.46 and P = 0.52 for ß1N11 and ß1C11 vs. WT mß1, respectively). Fitting both PO-V and logPO-V using Eq. 2 (Fig. 8, C and F; Table II), it was found that the major effect of the mß1 mutations is a reduced leftward shift of VhO. This is from –61-mV shift for wild-type mß1 to a –20-mV shift for ß1N11, and complete elimination in ß1C11. ß1N11 and ß1C11 reduced L0, compared with alone (Fig. 8, B and E; 5.5 e–3 and 8 e–3, respectively, relative to 9 e–2 for ), but to a somewhat lesser extent compared with wild-type ß1 (1.8 e–3). In summary, these results suggest that the intracellular domains are required for ß1 subunit effects on voltage sensor activation and explains why ß1N11 and ß1C11 do not negatively shift the G-V relationship (Fig. 7, D and H). In contrast, mutation of the intracellular domains has a much weaker effect on L0.
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subunit that perturbs ß1 effects on intrinsic opening and voltage sensor activation. This scenario predicts that deleting both intracellular domains should reconstitute ß1 subunit properties. We tested this possibility by generating ß1 mutations lacking both N- and C-terminal domains (ß1N10C11 and ß1N11C11). Coexpression of ß1 N10C11 with wild-type demonstrates that the double mutant, like the ß1N11 and ß1C11 mutants, eliminates the negative voltage shift of the G-V in high Ca2+ (Fig. 7 L). In addition, the ß1N10C11 mutant also perturbs the positive G-V shift in low Ca2+ (Fig. 7 L). These results suggest that the double deletion may also affect ß1's ability in modulating L0 and VhO. To directly examine effects of the double deletions on intrinsic and voltage-dependent gating, logPO-V relationship was obtained for BK/F315Y+ß1N10C11 using single channel recordings (Fig. 8 G). Fitting logPO-V relationship at limiting slope showed that unlike the single deletions, the ß1N10C11 significantly reduces voltage dependence of the closed to open equilibrium (zL is 0.13 ± 0.02 e0; Fig. 8 H). Analysis using Eq. 2 indicates that ß1N10C11 dramatically decreases ß1's reduction of L0 and eliminates ß1's ability to left shift VhO (Fig. 8 I; Table II). The above findings suggest that it is unlikely that ß1N11 and ß1C11 are dominant-negative mutations, and provides additional evidence that intracellular domains are required for stabilizing voltage sensor activation. Coexpression of wild-type and ß1N11C11 produced currents indistinguishable from BK/ alone (unpublished data). Although the protein was expressed (as assayed by immunohistochemistry; unpublished data), it appears that the conserved E11 residue is critical for coupling between and ß1 when the 10 and 11 residues of the N and the C terminus are deleted.
The ß1 subunit has the additional property of reducing the apparent voltage dependence (Q) of the conductance–voltage relationship. Intracellular domain chimeras (BK chimeras with related slo3 channels) that eliminate the negative shift of the G-V relationship do not affect the apparent voltage dependence (Qian et al., 2002). Similarly, we find that deletion of either intracellular domains and the double deletion, to an extent, still decrease Q (Fig. 7, D, H, and L). In combination with the double deletion effect on the V1/2 at low Ca2+ (Fig. 7 L), these results indicate that some effects by mß1 are retained by interactions in the transmembrane and/or extracellular domains.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Nimigean and Magleby, 1999; Lippiat et al., 2003; Orio and Latorre, 2005) and bovine ß1 (bß1) (Cox and Aldrich, 2000; Bao and Cox, 2005). Several of these studies also observed that below 1 µM Ca2+, ß1 either becomes less "effective" in shifting G-V relations (Meera et al., 1996; Cox and Aldrich, 2000; Nimigean and Magleby, 2000; Bao and Cox, 2005) or produces a positive shift in the G-V relationship (Orio and Latorre, 2005). Our studies with mß1 concur with the later, and indeed show a very large positive shift at submicromolar Ca2+.
How do ß1 subunits confer an increase in apparent Ca2+ sensitivity, and an increased slope for the V1/2–Ca2+ curve? By combining the F315Y limiting slope analysis with mutagenesis of the intracellular domain, we were able to uncover mechanisms that contribute to these properties. Utilization of the F315Y mutation with ß1 allowed us to directly measure the effect on PO by the negative shift of voltage sensor activation, as predicted by previous gating current measurements (Bao and Cox, 2005). The decrease in VhO and the negative shift of the G-V relationship are correlated in our mutations, indicating that effects on voltage sensor equilibrium by ß1 may be causal for the negative G-V shift, as predicted by Bao and Cox (2005). However, our simulations indicate that the negative G-V shift occurs equally across Ca2+ concentrations. This indicates that the increased slope of the V1/2–Ca2+ curve is not accounted for by effects on voltage sensor equilibrium. Rather, we found that ß1 decrease of intrinsic gating (L0) contributes to the increased slope of the V1/2–Ca2+ curve. Unlike VhO, the effect of L0 on V1/2 appears to be Ca2+ dependent where there is a greater positive shift of the G-V curve at low Ca2+ than high Ca2+. Surprisingly, it is this ß1 effect that reduces PO more so at low Ca2+ than at high calcium that gives a steeper Ca2+ response.
Previous studies had also inferred that human ß1 decreased BK channel's closed-to-open equilibrium (Orio and Latorre, 2005). However, this is somewhat controversial given that Bao et al. did not require a decreased closed to open equilibrium to explain bovine ß1 subunit effects (Bao and Cox, 2005). In part, this discrepancy may also be due to species differences. At 0 Ca2+, V1/2 for oocyte-expressed BK channels composed of mouse (mslo-mbr5; Butler et al., 1993) and bovine ß1 (Knaus et al., 1994) is 200 mV (Bao and Cox, 2005), and for BK channels composed of human and human ß1 expressed in oocytes is 250 mV (Orio and Latorre, 2005). In our study, mouse (Pallanck and Ganetzky, 1994) and mouse ß1 expressed in HEK293 cells resulted in an estimated V1/2 to be >300 mV. Thus, mouse (this study) and human ß1 subunits (Orio and Latorre, 2005) may have a greater effect on L0 than the bovine ß1 subunit (Bao and Cox, 2005). An additional variable is the expression system. Functional interaction between BK channel and ß subunits has been shown to be phosphorylation dependent (Erxleben et al., 2002; Jin et al., 2002). It is possible that similar to KCNQ channels (Nakajo and Kubo, 2005), BK channel phosphorylation status differs between oocytes (used in the previous studies) and HEK293 cells (used in this study).
ß1 and ß4 Subunits Share Similar Mechanisms
Interestingly, the major effects of ß4 are similar to the mouse ß1 subunit. Both cause a decrease in intrinsic opening and leftward voltage shifts for voltage sensor activation (Wang et al., 2006). The distinction is that the ß1 subunit has a crossover between inhibition and activation at low micromolar Ca2+ concentrations and is therefore generally regarded to promote channel activation. The ß4 subunit, in contrast, has a crossover at tens of micromolar Ca2+ concentration and is generally regarded to be a down-regulator for BK channels (Weiger et al., 2000; Brenner et al., 2005). It is indeed possible that quantitative differences in these two opposing effects, intrinsic gating or voltage sensor activation, underlie the distinction between ß1 and ß4 subunits.
ß1 Functional Domains
Finally, these studies contribute to our understanding of ß1 subunit domains that mediate interaction with BK channels. Previous studies using chimeras between ß1 and ß2 indicate that differences between these subunits can be ascribed to differences in the intracellular domains of the ß subunits (Orio and Latorre, 2005). Consistent with these studies, we find that most, but not all, of the effects of ß1 (effects on VhO and L0) are mediated by the intracellular domains. Predominant effects of the extracellular and transmembrane domain appear to be its influence on the equivalent gating charge conferred by ß1 subunits, and also a small effect on L0. An intriguing possibility may be that the intracellular domains of the ß1 subunit directly interact with the voltage sensor domain to modulate channel activation. Indeed, the recent finding that residues in S2 and S3, in addition to the S4 transmembrane domains, contribute to voltage sensor equilibrium (Horrigan and Aldrich, 2002; Ma et al., 2006) present the possibilities that ß1 intracellular domains may be tugging on any of the respective intracellular loops for S2–S4 to mediate effects on VhO.
However, other studies have found that perturbing the subunit N-terminal extracellular domain and the first transmembrane (S0) domains also has a profound effect on the negative shift of the G-V relationship conferred by ß1 (Wallner et al., 1996; Morrow et al., 2006). We cannot rule out the possibility that intracellular domains and transmembrane/extracellular domains of ß1 are allosterically coupled so that mutations in either domain perturb ß1 subunit effects. Alternatively, mutations in the extracellular domain of and intracellular domains of ß1 affect different aspects of BK channel gating that appear qualitatively similar if measured by the net effect of the G-V relationship. In this regard, future studies using the F315Y limiting slope analysis should provide a more accurate mapping of and ß1 subunit functional domains.
F315Y Provides a Useful Reagent for Measuring BK Channel Properties at the Limiting Slope
Historically, a number of other ion channel mutations have served to uncover mechanisms that would otherwise be difficult or not possible to resolve. One example is the ILT Shaker mutation. By separating the final open transitions from charge movement steps (Smith-Maxwell et al., 1998), the ILT mutation allowed biophysical studies to probe channel gating mechanisms (del Camino et al., 2005; Pathak et al., 2005). As well, the W434F mutation of Shaker channel blocks potassium conductance and facilitates gating current measurements (Perozo et al., 1993). Yet, as useful as these mutations are, they have their own caveats with regard to how they affect other channel gating properties. For example, W434F, in addition to blocking channel conductance, it also retains channels in a c-type inactivated state (Yang et al., 1997). This begs the question of how the F315Y mutation affects our ability to infer ß1 modulation of gating.
The F315Y mutation is located in the C-terminal residues of the S6 domain, a region that is ascribed to serving as the gate for Kv channels (Swartz, 2005). Our observations were that the F315Y had two effects. Most dramatic was an increase in intrinsic gating that is apparent as a large (30-fold) increase in open channel dwell times (Fig. 4; 11 ± 2 ms F315Y vs. 0.36 ± 0.02 ms WT at –60 mV, 5 nM Ca2+) and 10,000-fold increase in limiting slope PO (Fig. 4 D). As well, fitting to the HA model indicates a negative shift of voltage sensor activation of closed channels (VhC, Table II), perhaps indicating a change in channel conformation in the closed state. Taken together, a simplistic hypothesis is that the F315Y mutation destabilizes the closed gate. Thus, although F315Y may not be useful in reporting effects on VhC, several lines of evidence suggest that other F315Y and ß1 properties are qualitatively additive, indicating that their mechanisms are independent and not masked. Compared with wild-type BK/ channels, BK/+ß1 and BK/F315Y both display increased mean burst duration (Fig. 3 B; Nimigean and Magleby, 1999). Despite the dramatically increased burst durations of F315Y, this property of ß1 is conserved in the F315Y background (Fig. 5 D; F315Y+ß1 is 334 ± 12 ms vs. 11 ± 2 ms F315Y alone at –60 mV, 5 nM Ca2+). In addition, ß1 subunits confer a reduction in L0 in the F315Y background despite the large increase in intrinsic gating (L0) by the mutation. Other properties of ß1 also appear to be qualitatively retained, including the negative shift of open channel voltage sensor activation previously reported by Bao and Cox (2005). Thus, in many aspects, F315Y has effectively uncovered ß1-mediated modulation of BK channels.
With regard to estimating VhC, it is not clear if the F315Y mutation reports ß1 effects. Bao and Cox saw that bß1 conferred similar shifts of both VhO (–61 mV) and VhC (–71 mV). Our estimates of mß1 were an unequal shift of VhO (–61 mV) and VhC (–20 mV) in the F315Y background. The fact that F315Y alone has a VhC (+110 mV, Table II) that is quite different than wild-type subunits (+202 mV) creates the possibility that the F315Y mutation perturbs ß1 effects on VhC.
In conclusion, the increase in PO by the F315Y mutation has uncovered properties that were predicted by gating current measurements, and novel properties such as effects on intrinsic gating that were previously difficult to measure. One can predict that the mutation should continue to provide a valuable tool to identify critical residues that bridge functional interactions between the BK channel and ß1 subunits.
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ACKNOWLEDGMENTS |
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This work was supported by a National American Heart Association grant 0335007N and Sandler Program For Asthma Research to R. Brenner. B. Wang is supported by National Institutes of Health T32 training grant HL04776-23.
Olaf S. Andersen served as editor.
Submitted: 15 June 2006
Accepted: 3 November 2006
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