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Correspondence to Frank T. Horrigan: horrigan{at}mail.med.upenn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca2+ dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases IK and shifts the GK–V relation to more negative voltages, whereas MTSES and NEM shift the GK–V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] and voltage were also identified where Po is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca2+-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K+ conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the GK–V relation by >–80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.
Key Words: calcium • potassium channel • BK channel • cysteine • RCK domain
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit of BK channels contains a core domain resembling that of other voltage-dependent K+ channels with six transmembrane segments (S1–S6) including a charge S4 voltage sensor (Adelman et al., 1992
; Butler et al., 1993). In addition, the channel contains a cytosolic COOH-terminal tail domain that represents almost 70% of the channel protein as well as a unique NH2-terminal S0 transmembrane segment (Meera et al., 1997). Regions within the tail domain have been identified that influence channel activation by micromolar Ca2+ (Schreiber and Salkoff, 1997; Schreiber et al., 1999; Bao et al., 2002, 2004; Qian et al., 2002; Xia et al., 2002) as well as many other aspects of channel function under physiological or pathophysiological conditions, including regulation by millimolar Ca2+ or Mg2+ (Shi et al., 2002; Xia et al., 2002), protons (Avdonin et al., 2003), Heam-binding (Tang et al., 2003), phosphorylation (Schubert and Nelson, 2001), and oxidation (Tang et al., 2001, 2004). Although considerable progress has been made in identifying functional elements within the BK channel tail, many features of the structure and function of this very large domain remain unknown. Even where important regions have been characterized, it is often unclear how they interact with each other or the transmembrane core to influence channel activation. An important aspect of the mechanisms by which tail domain elements influence function must be their eventual coupling to channel opening, a linkage that potentially involves multiple protein domains and interactions, including effects on voltage- or Ca2+-dependent gating. Thus, it is likely that the tail domain contains not only regulatory elements that interact directly with signaling molecules, but also regions that are critical for coupling the action of these regulatory elements to the channel gate.
Two RCK (regulator of K+ conductance) homology domains (RCK1 and RCK2) have been identified within the NH2-terminal half of the BK channel tail, encompassing several functionally important regions (Jiang et al., 2002; Qian et al., 2002; Tang et al., 2004). The crystal structure of RCK domains are known for several prokaryotic proteins, including the Ca2+-dependent MthK channel (Jiang et al., 2001, 2002), providing a potential model of BK channel structure and function. By analogy with MthK, conformational changes in the BK channel RCK domains are proposed to be coupled to the pore through the S6–RCK1 linker (Jiang et al., 2002). Effects of linker modification on BK channel gating appear consistent with this hypothesis (Niu et al., 2004). However, the RCK domains of BK and MthK channels share <20% sequence identity, suggesting that their structures are not identical and that important differences may exist regarding interactions that occur within the RCK domain and with the rest of the channel protein. For example, a low-affinity Mg2+/Ca2+ binding site has been identified in the RCK1 domain that is not present in MthK (Shi et al., 2002; Xia et al., 2002). In addition, Mg2+-dependent activation of BK channels is inhibited by mutations in the S4 and S4–S5 linker, suggesting that interactions exist between the RCK1 domain and voltage sensor, whereas MThK is a two transmembrane channel that has no voltage sensor (Hu et al., 2003).
In the present study, we probed the function of the tail domain by studying changes in BK channel gating produced by various cysteine-modifying reagents (MTSES, MTSET, and NEM) applied to the intracellular side of the channel. Of the 30 cysteine residues present in the mSlo1 BK channel -subunit, only 3 are in the core domain, and these are located in extracellular regions of the NH2 terminus, S1–S2 linker, and pore loop. The majority of cysteines (24) are scattered throughout the tail domain and three more are located in the intracellular S0–S1 linker. Cysteine modification by thiol-specific MTS reagents or oxidation has been shown to inhibit BK channel activity (DiChiara and Reinhart, 1997; Tang et al., 2001, 2004; Erxleben et al., 2002). However, the functional changes and cysteines that underlie these changes have been only partially characterized.
Here, we determine the effects of cysteine modification on steady-state and kinetic properties of IK over a wide range of conditions to determine the impact of modification on voltage- and Ca2+-dependent gating as well as the relative stability of the closed and open conformation. BK channel gating is well described in terms of allosteric mechanisms where the closed–open conformational change does not require Ca2+ binding or voltage sensor activation but is promoted by either (Cox et al., 1997a; Horrigan and Aldrich, 1999, 2002; Horrigan et al., 1999; Rothberg and Magleby, 1999, 2000; Cui and Aldrich, 2000). By measuring steady-state activation under conditions that include 0 Ca2+ and extreme negative voltages, we can isolate effects on the closed–open transition from those on Ca2+- or voltage-dependent gating mechanisms. Moreover, we can distinguish whether perturbations to Ca2+- or voltage-dependent gating reflect changes in the activation of Ca2+ or voltage sensors or a change in the coupling of these sensors to channel opening (Horrigan and Aldrich, 2002).
By characterizing in detail the changes in BK channel gating produced by cysteine modification, we seek not only to identify important regions of the tail domain but also to understand the mechanisms by which these regions influence channel function. C911 has been identified previously in hSlo1 as a key cysteine whose modification is largely responsible for inhibitory effects of cysteine oxidation or MTSEA on BK channel activation (Tang et al., 2004). Modification of C911 is thought to inhibit Ca2+ binding to the nearby Ca2+-bowl domain (Schreiber and Salkoff, 1997). However, the effects on channel gating of cysteine-modifying reagents in our experiments differ from those described by Tang et al. (2004), and involve additional sites of action. Thus, we find that modification of C615 by MTSET also reduces apparent Ca2+ affinity, suggesting that the Heam-binding site, which includes C615, may influence Ca2+ binding. In addition, we find that modification of C430 in the RCK1 domain alters the relative stability of closed and open conformations as well as the coupling of both voltage sensor activation and Ca2+ binding to channel opening. The impact of C430 is particularly interesting because it lies in a region of the RCK domain that is not present in MthK. The effects on gating of C430 modification, or deletion of a region including C430, are discussed in light of known features of RCK structure and the recent proposal that the BK channel RCK domain exhibits spring-like mechanical properties (Niu et al., 2004).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) provided by L. Salkoff (Washington University, St. Louis, MO) in a BlueScript vector (Stratagene), or human homologue hSlo1 (hbr1) (Tseng-Crank et al., 1994) provided by T. Hoshi (University of Pennsylvania, Philadelphia, PA) in pCI-neo vector (Promega). hSlo1 point mutants (C348A, C422A, C430A, C615S, C628S, C630S, and C911A) and C(1–13)A, C(18–29)A, and C(1–13)AC(18–29)A constructs were also provided by T. Hoshi (Tang et al., 2001; Avdonin et al., 2003). For constructs containing multiple C to A mutations, the numbers in parentheses refer to sequentially numbered cysteines where the actual position in hSlo1 or mSlo1 are 14, 53, 54, 56, 141, 277, 348, 422, 430, 485, 498, 554, 557, 612, 615, 628, 630, 695, 722, 797, 800, 820, 911, 975, 995, 1001, 1011, 1028, and 1051. The hSlo1-
D construct in which eight amino acids were deleted (428–435) was prepared by overlap extension PCR, subcloned into full-length hSlo1-pCI-neo, and verified by sequencing. cRNA for mSlo1 was synthesized from BamH1-linearized cDNA using T3 polymerase and for hSlo1 and hSlo1 mutants from Not1-linearized cDNA using T7 polymerase. Xenopus oocytes were injected with 
0.5–5 ng of cRNA 4–20 d before recording and maintained at room temperature.
Electrophysiology
Currents were recorded using the patch clamp technique in the inside out configuration (Hamill et al., 1981). Upon excision, patches were washed with at least 20x volumes of internal solution. K+ currents were recorded with internal solutions containing (in mM) 110 KMeSO3 and 20 HEPES and an external (pipette) solution containing 100 KMeSO3, 10 KCl, 2 MgCl2, 20 HEPES. Internal solutions contained 40 µM (+)-18-crown-6-tetracarboxylic acid to chelate contaminant Ba2+ (Diaz et al., 1996; Neyton, 1996; Cox et al., 1997b). "0 Ca2+" solutions contained 2 mM EGTA reducing free Ca2+ to an estimated 0.8 nM based on the presence of 10 µM contaminant Ca2+ (Cox et al., 1997b). Ca2+ solutions were buffered with 5 mM HEDTA, and free Ca2+ was measured with a Ca2+ electrode (Orion Research Inc.). Nominal [Ca2+] reported as 1, 5, 10, 20, and 70 µM correspond to measured concentrations of 1.3, 4.4, 9.9, 17, and 66 µM, respectively. Ca2+ was added as CaCl2, and [Cl–] was adjusted to 10 mM with HCl. The pH of all solutions was 7.2. Solutions were prepared and experiments performed at room temperature (22–24°C).
Electrodes were pulled from thick-walled 1010 glass (World Precision Instruments), coated with wax (KERR sticky wax) and fire polished before use. Pipette access resistance measured in the bath solution (0.8–1.5 M
) was used as an estimate of series resistance (Rs) to correct the voltage at which IK was recorded. The corrected voltage was used in determining membrane conductance (GK) from tail current measurements and in plotting the voltage dependence of GK. Series resistance error was <15 mV for all data presented.
Data were acquired with an Axopatch 200B amplifier (Axon Instruments) set in patch mode with the amplifier's internal 4-pole Bessel filter set at 100 kHz. Currents were subsequently filtered by an 8-pole Bessel filter (Frequency Devices Inc.) at 20 kHz and sampled at 100 kHz with an 18 bit A/D converter (Instrutech ITC-18). A P/4 protocol was used for leak subtraction (Armstrong and Bezanilla, 1974) with a holding potential of –80 mV. A Macintosh-based computer system was used in combination with Pulse Control acquisition software (Herrington and Bookman, 1995) and Igor Pro for graphing and data analysis (WaveMetrics Inc.). A Levenberg-Marquardt algorithm was used to perform nonlinear least-squared fits.
Under conditions where the open probability (PO) is small (<10–3), unitary currents were observed in patches containing hundreds of channels, and NPO was determined from steady-state recordings of 1–60 s duration that were digitally filtered at 5 kHz. NPO was determined from all-points amplitude histograms by measuring the fraction of time spent (Pk) at each open level (k) using a half-amplitude criteria and summing their contributions NPo=
kPk. Po was determined by combining NPO measurements with an estimate of N = GKmax/gK, where GKmax was determined by fitting a Boltzmann function (GK = GKmax[1 + exp(–ze(V – Vh)/kT)]–1) to the macroscopic GK–V relation in the same patch, and gK is the single channel conductance.
Patch to patch variations in the half-activation voltage of GK–V relations are observed for mSlo1 (Horrigan et al., 1999) and hSlo1 (Stefani et al., 1997), possibly due to differences in the redox state of channels (DiChiara and Reinhart, 1997; Tang et al., 2001). Such variation causes broadening in averaged voltage-dependent relationships relative to individual experiments. To compensate for this effect, Vh was determined for each patch and compared with the mean for all experiments (<Vh>) at the same [Ca2+]. GK–V relations from individual experiments were then shifted along the voltage axis to <Vh> before averaging. This procedure yields average relations that more accurately represent the shape of individual GK–Vs. To determine average GK–V relations for channels modified by MTSES, MTSET, or NEM, the GK–V shift produced by steady-state modification Vh = Vh (modified) – Vh (control) was determined from individual experiments, and the modified GK–Vs in each [Ca2+] were shifted to a mean Vh (<Vh(modified)> = <Vh control> + <Vh>) before averaging. In this way, the relative position of mean GK–V relations and Vh–[Ca2+] relations before and after modification accurately reflect the mean G–V shift (<Vh>) rather than patch to patch differences in Vh for control and modified GK–Vs that were averaged from different experiments.
Reagents
MTSET ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide) and MTSES (sodium (2-sulfanatoethyl) methanethiosulfonate) were obtained from Toronto Research Chemicals, dissolved in water at 100 mM and 1 M, respectively, and stored at –20°C. During experiments, aliquots were thawed and stored on ice for not longer than 30 min before diluting 1,000-fold into the appropriate internal solution just before application. NEM (N-ethyl maleimide; Sigma-Aldrich) was prepared at 250 mM in internal solutions containing different Ca2+ and stored at 4°C before diluting into the same internal solution at 20 mM immediately before use. Solution exchange and reagent applications were done manually within 5 s by exchanging the bath solution (200 µl) with 5–20 volumes of internal solution.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tang et al., 2001). Therefore, we typically waited 30 min following patch excision to allow the GK–V relation to stabilize, before acquiring control records and applying cysteine-modifying reagents. While this procedure may allow some cysteines to become oxidized, it assures that effects of cysteine-modifying reagents were studied in the virtual absence of spontaneous changes in gating. During reagent application, the membrane was held at –80 mV, and macroscopic potassium currents (IK) were evoked by brief 25-ms test pulses every 10 s to a potential near the half-activation voltage (Vh). Fig. 1 (A–C) shows the effect on IK of 1 mM MTSES, 100 µM MTSET, and 20 mM NEM, respectively. MTSES, applied in the absence of Ca2+, produced a gradual decrease in both IK evoked at +160 mV and tail current following the test pulse (Fig. 1 A). An approximate 60% steady-state decrease in current amplitude was observed after 250 s. By contrast, MTSET in 0 Ca2+ produced a rapid twofold increase in IK within the 10-s interval between test pulses (Fig. 1 B). NEM, like MTSES, produced a gradual decrease in IK, achieving an 80% steady-state reduction in 300 s in the presence of 5 µM Ca2+ (Fig. 1 C).
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; Morales et al., 1996); and 1 mM NaCl reproduces the effect of 1 mM Na-MTSES on the I–V relation (Fig. 1 D). To minimize the influence of channel block on analysis of cysteine modification, concentrations of MTSET and MTSES were limited to 100 µM and 1 mM, respectively. In addition, the time course of modification and conductance–voltage (GK–V) relations were determined by measuring tail currents at a voltage (–80 mV) where block is negligible. Finally, reagent was washed out after steady-state modification when possible so that IK could be compared before and after modification in the absence of reagent. GK–V relations obtained in this way before and after modification by MTSES (Fig. 1 F), MTSET (Fig. 1 G), and NEM (Fig. 1 H) indicate that changes in IK amplitude observed in Fig. 1 (A–C) reflect a shift of the GK–V along the voltage axis and, for MTSES and NEM, a decrease in GKmax estimated from fits to a Boltzmann function.
Effects of Cysteine Modification are Ca2+ Dependent
Because BK channel activation is Ca2+ dependent, we examined the effects of applying cysteine-modifying reagents in different [Ca2+]i, from 0 to 70 µM. At each [Ca2+]i, test pulse voltage was less than or equal to Vh such that IK should change if the GK–V relation is altered. However, marked differences in the extent to which IK changed were observed in different [Ca2+]i (Fig. 2).
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= 61 s). In 5 µM Ca2+, GK decreased by <10% over the same time period. This small slow decrease cannot be distinguished from the spontaneous rundown of GK that sometimes occurs in the absence of reagent presumably due to channel oxidation. A similar failure of MTSES to alter GK was observed at all [Ca2+]i >1 µM (unpublished data).
MTSET and NEM, unlike MTSES, significantly altered GK in 5 µM Ca2+. 100 µM MTSET increased GK by 50% with a time course, as in 0 Ca2+, that was too rapid to resolve (Fig. 2 D). 20 mM NEM decreased GK by >80% with an exponential time course ( = 127 s; Fig. 2 F). However, conditions were also identified where changes in GK produced by MTSET or NEM are small. Surprisingly, MTSET did not alter GK in 1 µM Ca2+ (Fig. 2, C and D), although increases were observed in both higher (5 µM) and lower (0) [Ca2+]i (Fig. 2 D). Conversely, the effect of NEM on GK in 0 Ca2+ (Fig. 2, E and F) or 70 µM Ca2+ (Fig. 2 F) was much less than that observed in 5 µM Ca2+.
Cysteine Modification is Not Prevented by Ca2+ Binding
The results in Fig. 2 demonstrate that interaction among cysteine-modifying reagents, Ca2+, and BK channel gating is complex. Not only are the effects of MTSES, MTSET, and NEM on GK Ca2+ dependent, but the pattern of Ca2+ dependence for each reagent is different. To understand these results, it's important to distinguish between channel modification and the modification effect. Modification refers to the reaction of reagent with the BK channel protein, most likely forming a covalent bond with one or more cysteine residues. The modification effect is the change in channel function produced by cysteine modification. Although a modification effect indicates that cysteines have been modified, the converse is not necessarily true. That is, failure to observe a modification effect does not indicate a lack of cysteine modification if modification has no effect on function or produces complex changes in channel gating that are difficult to detect.
Two mechanisms could account for the failure to observe a modification effect at certain [Ca2+]i in Fig. 2. First, the accessibility of cysteine residues to reagent might be state dependent such that the rate of modification is influenced by Ca2+ binding. That is, modification of key cysteines might be inhibited by the presence or absence of Ca2+. Second, cysteine modification might alter the Ca2+ sensitivity of channel gating such that the change in GK produced by modification at each [Ca2+]i is different. Experiments shown in Fig. 3 suggest that the latter mechanism accounts for the results in Fig. 2.
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Fig. 3 A plots G–V relations in 20 and 70 µM Ca2+ before and after application of NEM. The corresponding sequence of solution changes and Vh measurements are indicated in Fig. 3 B. G–Vs were initially measured in 20 and 70 µM Ca2+, and then 20 mM NEM was applied for 300 s in the presence of 70 µM Ca2+, producing no change in Vh (Fig. 3 B) or GK (Fig. 2 F). G–Vs obtained in 70 µM Ca2+ before application of NEM and after washout are indistinguishable (Fig. 3 A). However, the G–V in 20 µM Ca2+ measured after washout of NEM was shifted by –16 mV relative to the control, similar to a –22 ± 5 mV shift (mean ± SEM) observed when NEM was applied in the presence of 20 µM Ca2+. Thus NEM in 70 µM Ca2+ does modify cysteines and alter channel gating despite failure to alter steady-state activation in 70 µM Ca2+.
Fig. 3 (C and D) plots GK in 5 µM Ca2+ as MTSES is applied for 200 s before applying NEM or MTSET, respectively. When applied individually in 5 µM Ca2+, MTSES has no effect on GK, whereas NEM decreases GK by >80% (Fig. 2 F; Fig. 3 C, line) and MTSET increases GK by 50% (Fig. 2 D; Fig. 3 D, line). However, MTSES pretreatment prevents the effects of NEM and MTSET (Fig. 3, C and D, symbols). This result indicates that cysteines are modified by MTSES in 5 µM Ca2+ despite the failure to alter GK. Moreover, MTSES must modify the cysteines that underlie the modification effects of MTSET and NEM. This conclusion is supported by observations that MTSET pretreatment prevents the modification effects of NEM and MTSES (unpublished data). Thus, all three reagents appear to modify the same cysteine residues.
Effects of Cysteine Modification on IK Kinetics
Changes in steady-state activation produced by cysteine modification are accompanied by changes in IK kinetics (Fig. 4). Comparison of normalized currents evoked by voltage pulses before and after MTSES treatment in 0 Ca2+ (Fig. 4 A) shows that modification slows IK activation at +240 mV 2.4-fold but has no effect on tail current decay at –80 mV. This is also evident in Fig. 4 B, which plots the time constants of IK relaxation ((IK)) at different voltages in 0, 5, and 70 µM Ca2+ before and after steady-state modification. In 0 Ca2+, (IK) is increased at V 
+200 mV but is unchanged from +240 to +180 mV. That (IK) increases only at the most positive voltages suggests that cysteine modification slows the rate-limiting forward transitions from closed to open (activation) without altering the reverse transition (deactivation), consistent with a decrease in open probability and shift of the G–V to more positive voltages in 0 Ca2+ (Fig. 1 F). Likewise, (IK) is not altered in 70 µM Ca2+ (Fig. 4 B), consistent with the lack of a G–V shift in high [Ca2+]. However, in 5 µM Ca2+, activation is slowed (Fig. 4, A and B), although IK amplitude is unchanged (Fig. 2 B; Fig. 3, C and D). This result provides additional evidence that channels are modified even when steady-state activation is unchanged, and suggests that MTSES does not act merely to alter the rate-limiting transition.
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(IK)–V relationships in 0, 5, or 70 µM Ca2+ (Fig. 4 D) exhibit similar responses to MTSET, increasing at voltages more negative than peak (IK) and unchanged at the most positive voltages. A slowing of deactivation is consistent with the increase in steady-state activation observed at these [Ca2+]. Likewise, in 1 µM Ca2+, no change in either IK kinetics (Fig. 4 C) or amplitude (Fig. 2 D) is observed.
Effects of NEM on the (IK)–V relation were not examined. However, the response to test pulses in 0, 10, and 70 µM Ca2+ (Fig. 4 E) reveals a slowing of IK activation with little effect on tail current decay, similar to the effect of MTSES and consistent with the decrease in steady-state activation produced by NEM (Fig. 2 F).
Cysteine Modification Alters the Ca2+ Dependence of Steady-state Activation
The above results indicate that cysteines are modified even at [Ca2+]i where no change in steady-state activation is evident. Therefore the apparent Ca2+ sensitivity of MTSES, MTSET, and NEM action (Figs. 1 and 2) must reflect effects of cysteine modification on the Ca2+ dependence of channel gating rather than effects of Ca2+ binding on cysteine accessibility. To further characterize changes in Ca2+-dependent gating, we determined GK–V relations at many [Ca2+]i before and after steady-state modification (Figs. 5 and 6). In these experiments, control data were recorded at various [Ca2+]i, and then cysteine-modifying reagents were applied at [Ca2+]i where the extent of modification could be monitored, as in Fig. 1, until a steady-state current amplitude was attained. Subsequently, the reagent was washed out and G–Vs for the modified channels were determined in different [Ca2+]i.
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; Horrigan and Aldrich, 2002). However, in contrast to previous studies, Vh is 60 mV more positive at all [Ca2+]i, and the estimated GKmax decreases at low Ca2+ (<5 µM), rather than remains constant. These two differences may be related and probably reflect that membrane patches in the current study were excised for an extended period (30 min) before G–Vs were recorded.
That GKmax appears to decrease in low Ca2+ is relevant to our analysis because estimation of Vh is dependent on the value of GKmax used to fit the GK–V relation. If GKmax is not constant, the Vh–[Ca2+] relation will be influenced by our ability to estimate GKmax at each [Ca2+]. In high [Ca2+] (>5 µM), GKmax was usually measured directly from the saturation of GK at positive voltages. In 1–5 µM Ca2+, examples exist where GK was observed to saturate at voltages approaching +300 mV (e.g., Fig. 5 B). However, GKmax at these [Ca2+] was more typically estimated by fitting a nearly saturating G–V and allowing all parameters to vary freely as in Fig. 5 A. In this way, GKmax in 1 µM was estimated to be 66 ± 12% (mean ± SEM) of that in 70 µM Ca2+. For [Ca2+] 
1 µM, GKmax could not be reliably estimated and was therefore approximated by the value determined in 1 µM Ca2+ (e.g., Fig. 5 A). GK–V relations fit in this way from different experiments were normalized to GKmax and averaged to illustrate the effect of Ca2+ on Vh (Fig. 6 A). As in previous studies, the normalized G–V shifts along the voltage axis in response to Ca2+ with little change in shape. The mean Vh–[Ca2+] relation obtained from these data (filled symbols, Fig. 5 C) is also similar in shape to that obtained from a previous study (open symbols) (Horrigan and Aldrich, 2002) but is shifted to more positive voltages.
The effects of cysteine-modifying reagents on the Ca2+ dependence of steady-state activation are shown in Fig. 6. Normalized G–V relations (mean ± SEM) obtained following modification by MTSES (Fig. 6 B), MTSET (Fig. 6 C), or NEM (Fig. 6 D) are similar in shape to the control (Fig. 6 A). The apparent charge (z) determined from Boltzmann fits to these data differ only at Ca2+ < 1 µM (Fig. 6 E). However, comparison of Vh–[Ca2+] relations for control and modified channels indicate that each reagent has a distinct effect on the Ca2+ dependence of steady-state activation (Fig. 6, E–G). MTSES increases Vh at low Ca2+ (1 µM) but not high Ca2+ (Fig. 6 E). MTSET fails to alter Vh over a range of [Ca2+] from 0.5 to 2 µM but decreases Vh at both higher and lower [Ca2+] (Fig. 6 E). NEM appears to shift the Vh–[Ca2+] relation along the Ca2+ axis such that Vh increases over a wide range of intermediate [Ca2+] but is unchanged in 0 Ca2+ or 70 µM Ca2+.
Effects of Cysteine Modification on Steady-state Activation at Extreme Negative Voltages
The complex effects of MTSES, MTSET, and NEM on the Vh–[Ca2+] relation suggest that cysteine modification alters multiple features of BK channel function. Shifts in the G–V relation may be caused by changes in either Ca2+- or voltage-dependent gating or the energetics of channel opening (Cox et al., 1997a; Cui and Aldrich, 2000; Horrigan and Aldrich, 2002). However, changes in any one of these processes cannot account for the different effects on Vh produced by these reagents. To better determine how each aspect of gating is altered by cysteine modification, we examined steady-state activation at extreme negative voltages (Fig. 7).
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). This phenomena is evident as a weakly voltage-dependent plateau in the PO–V relation at V < –80 mV when PO is plotted on a log scale versus voltage. Control PO–V relations (mean ± SEM) 0 Ca2+ and 70 µM Ca2+ are plotted in this way in Fig. 7 A. In 0 Ca2+, PO at –120 mV is very small (10–7), representing the closed–open equilibrium in the absence of Ca2+ binding or voltage sensor activation. However, PO(–120) increases by almost four orders of magnitude in 70 µM Ca2+ (Fig. 7 A), reflecting the strong interaction between Ca2+ binding and channel opening. Thus, PO at extreme negative voltages provides information about Ca2+-dependent gating and the energetics of channel opening in isolation from voltage-dependent gating. The effects of MTSET on PO at extreme negative voltages are surprisingly different than those at more positive voltages. PO–V relations in 0, 5, and 70 µM Ca2+ all increase at positive voltages in response to MTSET (Fig. 7 B), corresponding to approximate 40-mV decreases in Vh (Fig. 6 G). However, at –120 mV, PO decreases in 5 µM Ca2+, increases in 70 µM Ca2+, and is relatively unchanged in 0 Ca2+ (Fig. 7, B and C). That the effects of MTSET on Po at positive and negative voltages are different imply that cysteine modification alters voltage-dependent gating. That MTSET changes PO(–120) in a Ca2+-dependent manner indicates that Ca2+-dependent gating is also perturbed. However, failure to change PO(–120) in 0 Ca2+ suggests that MTSET does not merely alter the energetics of channel opening.
To characterize the changes in gating that occur in the absence of voltage sensor activation, we determined the ratio Rmod = PO(modified)/PO(control) at –120 mV in various [Ca2+] for MTSET, MTSES, and NEM. Steady-state currents were recorded at negative voltages before and after modification from macro-patches containing several hundred channels. Because PO is small at –120 mV, single channel currents are observed (Fig. 7 C) and NPo was determined from amplitude histograms (Fig. 7 D, see MATERIALS AND METHODS). In most cases, measurements were made at several voltages from –160 to –80 mV to confirm that NPo is weakly voltage dependent at –120 mV. However, IK at more positive voltages was often too large to record. Therefore, the number of channels in the patch (N) was not routinely determined, and Rmod was evaluated as NPO(modified)/NPO(control). To limit potential contributions of channel rundown to this ratio (e.g., a change in N), Rmod in each patch was determined at a single [Ca2+], immediately before and after steady-state modification.
Rmod determined from many different patches (mean ± SEM) is plotted versus [Ca2+] for MTSET (Fig. 7 E), MTSES (Fig. 7 F), and NEM (Fig. 7 G). In 0 Ca2+, Rmod = 1 for MTSET, indicating that PO(–120 mV) is unchanged. However, for MTSES and NEM, Rmod = 0.2, indicating that these reagents, unlike MTSET, inhibit channel opening in the absence of voltage sensor activation or Ca2+ binding. All three reagents also cause a decrease in PO(–120 mV) at intermediate [Ca2+] from 1–10 µM (i.e., Rmod < 1). However, in higher [Ca2+], PO is increased by MTSET and to a lesser extent by NEM but is relatively unchanged by MTSES.
Although each reagent has distinct effects on PO (–120 mV), they all enhance the response to Ca2+. That is, the Ca2+-dependent change in PO from 0 Ca2+ to 70 µM Ca2+ at negative voltages is increased by cysteine modification. This effect is evident for MTSET from the Po–V relations in Fig. 7 B. Moreover, that Rmod in 70 µM Ca2+ is always greater than Rmod in 0 Ca2+ indicates that this enhancement occurs for all three reagents (Fig. 7, E–G). Such an increase in the response to Ca2+ suggests that the coupling between Ca2+ binding and channel opening is strengthened by cysteine modification.
Identification of Modification-sensitive Cysteines
BK channel function is sensitive to modification of multiple cysteine residues (Tang et al., 2004). To identify these modification-sensitive residues, we examined the effects of MTSET, MTSES, and NEM on mutant constructs of hSlo1 that lack one or more cysteines. hSlo1 channels contain 29 cysteines, all of which are present in mSlo1. Aside from an additional 56–amino acid COOH-terminal sequence including one cysteine present in mSlo1, hSlo1 shares 99% sequence identity with mSlo1 and exhibits similar functional properties (Brenner et al., 2000; Tang et al., 2001). The response of hSlo1 to cysteine-modifying reagents was not as extensively characterized as mSlo1. However, we tested the effects of MTSES and MTSET under many of the conditions described above for mSlo1 and observed no appreciable difference in terms of the direction and magnitude of changes in Vh and PO(–120) (unpublished data).
Initially, we examined several constructs in which multiple cysteines were replaced by alanine. When all 29 cysteines are replaced, channels express poorly and are difficult to study (Tang et al., 2004). However constructs lacking either the first 13 (C(1–13)A) or last 12 cysteines (C(18–29)A) express macroscopic currents. We tested the effects of MTSET, MTSES, and NEM on these two constructs in 5 µM Ca2+. In both cases, GK was greatly altered by modifying reagents (Fig. 8, A and B), indicating that modification-sensitive cysteines remain. Moreover, a construct in which both sets of cysteines were replaced by alanine (C(1–13)A/C(18–29)A) expressed poorly but exhibited a clear decrease in open probability in response to NEM (Fig. 8 C).
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). We also examined the effect of MTSET on a point mutant C911A (Fig. 8 B) and observed a response similar to that of C(18–29)A. Thus, it is clear that modification of C911 cannot be solely responsible for the effects of MTSES and MTSET in the present study. Although mutants were affected by cysteine-modifying reagents, their response was not identical to the wild type (WT). C(1–13)A GK is decreased by NEM (Fig. 8 A), similar to the WT (Fig. 2 F). However MTSET decreases C(1–13)A GK as opposed to an increase for the WT (Fig. 2 D). In contrast, GK for C(18–29)A or C911A was immediately increased by MTSET (Fig. 8 B). However, this rapid increase was followed by a slow decay that is not observed with the WT. In addition, C(18–29)A GK is decreased by MTSES in 5 µM Ca2+, whereas WT GK at the same [Ca]i is unaffected by MTSES (Fig. 2 B).
Differences in the response of WT, C(1–13)A, and C(18–29)A channels to cysteine-modifying reagents suggest that some of the removed cysteines may be modification sensitive. However, these differences are difficult to characterize and interpret owing to changes in channel function produced by the mutations. Both C(1–13)A and C(18–29)A required higher voltages to activate than the WT (Tang et al., 2004) and therefore could not be studied in the absence of Ca2+. Given the complex interactions that occur between cysteine modification and channel function in the WT, it is possible that a change in gating caused by mutation could affect the response to cysteine modification. It is also possible that extensive mutation of cysteines could alter channel structure such that the accessibility of the remaining cysteines to modifying reagents is different than in the WT.
To minimize effects of mutation on channel structure or function, we examined the response to MTS reagents of several mutants where single cysteines were replaced. We focused on two regions in the COOH-terminal tail domain, the RCK1 domain and four central cysteines (C14-C17). The RCK1 domain is known to be functionally important and contains several cysteine residues, making it a likely candidate for modulation by cysteine modification. The response of C(1–13)A/C(18–29)A to NEM in Fig. 8 C suggests that at least one of the four remaining central cysteines may be modification sensitive in the WT. A cysteine residue was identified in each of these two regions that contribute significantly to the response to MTS reagents (Figs. 9 and 10).
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GK–V relations for C430A, in different [Ca2+] (Fig. 9 A), are similar to those of WT channels and exhibit similar Vh except at low [Ca2+] (<1 µM), where C430A is more difficult to activate and Vh is increased. The difference between Vh–[Ca2+] relations for WT and C430A (Fig. 9 B) resemble those produced by modification of the WT with MTSES (Fig. 6 F), suggesting that removal of C430 or its modification by MTSES may have similar effects on channel function. Consistent with the idea that MTSES acts primarily by modifying C430, C430A is almost insensitive to MTSES. In 0 Ca2+, MTSES decreases WT GK by 80% but has no detectable effect on C430A under the same conditions (Fig. 9 C). Similarly, MTSES had no significant effect on the Vh–[Ca2+] relation for C430A (Fig. 9 E). Likewise, MTSES produced little change in Po(–120) for C430A (i.e., Rmod 1; see Fig. 12 C).
Mutation of C430 reduces, but does not eliminate, effects of MTSET on channel gating. In 0 Ca2+, MTSET produces a rapid increase in WT IK, but C430A exhibits a slow decrease (Fig. 9 D). Vh for the mutant also increases slightly in 0 Ca2+ although no change in Vh is detected at higher [Ca2+] (Fig. 9 F). Although C430A almost abolishes the effects of MTSET on Vh, it does not eliminate changes in PO(–120) (Fig. 10 A). Similar to the WT, PO(–120) for C430A is unaffected by MTSET in 0 Ca2+ but is markedly decreased in 10 µM Ca2+ (Fig. 10 A). In contrast, MTSET has no effect on PO(–120) in 70 µM Ca2+ (Fig. 10 A), whereas the WT shows an approximate fivefold increase (Fig. 7 C). GK–V relations for C430A measured from individual patches immediately before and after treatment with MTSET confirm that there is little change in Vh in 10 or 70 µM Ca2+ and a small change in 0 Ca2+(Fig. 10 B).
The differences in the response of WT and C430A channels to MTSET suggest that modification of C430 by MTSET may act to both increase PO(–120 mV) in the presence of Ca2+ and shift the GK–V relations to more negative voltages. At intermediate [Ca2+], an increase in PO(–120) produced by modification of C430 could be masked by a decrease in PO(–120) produced by modification of an additional site. This hypothesis is supported by the observation that the decrease in PO(–120) produced by MTSET in 10 µM Ca2+ is at least twofold greater for C430A than for the WT (see Fig. 12 E).
C615 in the Heam-binding Site Is Sensitive to MTSET
The response of C(1–13)A/C(18–29)C to NEM (Fig. 7 C) suggests that at least one of four remaining cysteines in this construct (C612, C615, C628, C630) is also important for the effects of cysteine modification. We examined the effect of point mutations in three of these residues. Since the C430 site appears responsible in large part for changes in Vh and increases in PO(–120) in high Ca2+, we screened for the ability of MTSET to decrease PO(–120) in 10 µM Ca2+. C628S and C630S both exhibited substantial decreases in PO(–120) like the WT channel (unpublished data). However C615S, in the Heam-binding site, eliminated this effect of MTSET (Fig. 10 C, middle). Despite a decreased response to MTSET in 10 µM Ca2+, C615S still exhibits a marked increase in PO(–120) in 70 µM Ca2+ (Fig. 10 C, left). Similarly, Po–V relations in 1 and 5 µM Ca2+ were shifted to more negative voltages (Fig. 10 C, right). These results are consistent with the assumption that decreases in Vh and increases in PO(–120) in 70 µM Ca2+ reflect modification of C430 by MTSET.
Deletion of the RCK1 D–ßD Linker Enhances Channel Activation
The sensitivity of C430 to modification and mutation suggests that this residue and/or nearby residues in the RCK1 domain play important roles in channel gating. The RCK1 domain is thought to have a Rossmann-fold topology containing a series of alternating -helices and ß-sheets (Jiang et al., 2001). Alignment of BK channel sequences with RCK domains of known structure indicate that C430 is located between -helix D (D) and ß-sheet D (ßD) (Jiang et al., 2001, 2002). Fig. 11 A compares the sequences of mSlo1, hSlo1, and the fly homologue dSlo1 in this region with that of the MthK channel based on an alignment from Jiang et al. (2002) of BK channels with eight prokaryotic channels and transporters containing RCK domains. The D–ßD linker of MthK and other prokaryotic RCK domains contains only two amino acids. However BK channels contain an additional eight–amino acid domain including C430. The absence of this domain from the prokaryotic channels suggests it is not critical to RCK domain structure. Yet its sequence is highly conserved among BK channels, suggesting a possible functional role.
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], 0.6 [
], 1.3 [
], 4.4 [
], 10 [
], 17 [
], 31 [
], 51 [
], 66 [
]). For [Ca2+]< 1 µM, G–Vs were fit using GKmax determined from the 1 µM Ca2+ fit. (B) GK–V relations from another patch confirm that GK appears to saturate in 1 µM Ca2+ at a lower GKmax than in 10 µM Ca2+. (C) Vh–[Ca2+] relation from the present study (•) (mean ± SEM) is similar in shape to that from a previous study of mSlo1 (O) (Horrigan and Aldrich, 2002
]) obtained following steady-state modification by MTSES (B), MTSET (C), or NEM (D) are similar in shape to the control (A) but exhibit different half activation voltages. (E) Apparent charge (z) determined from Boltzmann fits to the mean G–Vs (lines, A–D) are plotted versus [Ca2+] (control [•], MTSES [
