An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating

Large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming alpha subunits and a family of tissue-specific accessory beta subunits. The smooth muscle-specific beta1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of beta1 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 beta1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca(2+) binding) due to the very low open probability in the presence of beta1. In this study, we used a mutation of the alpha 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 beta1 subunit. Effects of beta1 on steady-state open probabilities of both wild-type alpha and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse beta1 has two major effects on channel's gating energetics. beta1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca(2+). Further, P(O) measurements at limiting slope allow us to infer that beta1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca(2+) concentrations. Using the F315Y alpha subunit with deletion mutants of beta1, we also demonstrate that the small N- and C-terminal intracellular domains of beta1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that beta1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.     

Heme regulates allosteric activation of the Slo1 BK channel

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531-535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G-V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G-V simulated by weakening the coupling of both Ca(2+) binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca(2+)- and voltage-dependent gating as well as intrinsic stability of the open state.     

A role for the S0 transmembrane segment in voltage-dependent gating of BK channels

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channel's large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1-S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel alpha and beta subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance-voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channel's voltage sensor.     

Allosteric linkage between voltage and Ca(2+)-dependent activation of BK-type mslo1 K(+) channels

The activation of BK type Ca(2+)-activated K(+) channels depends on both voltage and Ca(2+). We studied three point mutations in the putative voltage sensor S4 or S4-S5 linker regions in the mslo1 BK channels to explore the relationship between voltage and Ca(2+) in activating the channel. These mutations reduced the steepness of the open probability - voltage (P(o) - V) relation and increased the shift of the P(o) - V relations on the voltage axis in response to increases in the calcium concentration. It is striking that these two effects were reciprocally related for all three mutations, despite different effects of the mutations on other aspects of the voltage dependence of channel gating. This reciprocal relationship suggests strongly that the free energy contributions to channel activation provided by voltage and by calcium binding are simply additive. We conclude that the Ca(2+) binding sites and the voltage sensors do not directly interact. Rather they both affect the mslo1 channel opening through an allosteric mechanism, by influencing the conformational change between the closed and open conformations. The mutations changed the channel's voltage dependence with little effect on its Ca(2+) affinitiy.     

Mg2+ enhances voltage sensor/gate coupling in BK channels

BK (Slo1) potassium channels are activated by millimolar intracellular Mg(2+) as well as micromolar Ca(2+) and membrane depolarization. Mg(2+) and Ca(2+) act in an approximately additive manner at different binding sites to shift the conductance-voltage (G(K)-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg(2+) action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca(2+). Evidence that Ca(2+) acts independently of voltage sensor activation includes an ability to increase open probability (P(O)) at extreme negative voltages where voltage sensors are in the resting state; 2 microM Ca(2+) increases P(O) more than 15-fold at -120 mV. However 10 mM Mg(2+), which has an effect on the G(K)-V relation similar to 2 microM Ca(2+), has no detectable effect on P(O) when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg(2+) when channels are closed, indicating that Mg(2+) does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg(2+) in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg(2+) increases P(O) only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg(2+) from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca(2+). The effects of Mg(2+) on steady-state activation and I(K) kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg(2+) and voltage sensor that may underlie this mechanism.     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Functional effects of auxiliary beta4-subunit on rat large-conductance Ca(2+)-activated K(+) channel

Large-conductance calcium-activated potassium (BK(Ca)) channels are composed of the pore-forming alpha-subunit and the auxiliary beta-subunits. The beta4-subunit is dominantly expressed in the mammalian central nervous system. To understand the physiological roles of the beta4-subunit on the BK(Ca) channel alpha-subunit (Slo), we isolated a full-length complementary DNA of rat beta4-subunit (rbeta4), expressed heterolgously in Xenopus oocytes, and investigated the detailed functional effects using electrophysiological means. When expressed together with rat Slo (rSlo), rbeta4 profoundly altered the gating characteristics of the Slo channel. At a given concentration of intracellular Ca(2+), rSlo/rbeta4 channels were more sensitive to transmembrane voltage changes. The activation and deactivation rates of macroscopic currents were decreased in a Ca(2+)-dependent manner. The channel activation by Ca(2+) became more cooperative by the coexpression of rbeta4. Single-channel recordings showed that the increased Hill coefficient for Ca(2+) was due to the changes in the open probability of the rSlo/rbeta4 channel. Single BK(Ca) channels composed of rSlo and rbeta4 also exhibited slower kinetics for steady-state gating compared with rSlo channels. Dwell times of both open and closed events were significantly increased. Because BK(Ca) channels are known to modulate neuroexcitability and the expression of the beta4-subunit is highly concentrated in certain subregions of brain, the electrophysiological properties of individual neurons should be affected profoundly by the expression of this second subunit.     

Mg(2+) binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

BK channels are activated by intracellular Ca(2+) and Mg(2+) as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca(2+) sites, one low-affinity Mg(2+) site, and a voltage sensor. This study further investigates the mechanism of Mg(2+) activation by using single-channel recording to determine separately the action of Mg(2+) on the open and closed states of the channel. To limit Mg(2+) action to the Mg(2+) sites, the two high-affinity Ca(2+) sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg(2+) decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg(2+) can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg(2+) action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (P(o)) is dramatically increased by 10 mM Mg(2+) when voltage sensors are activated with either depolarization or the mutation R210C. The increased P(o) arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg(2+) has no detectable effects on P(o) or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg(2+) can bind to and alter the gating of both closed and open states to increase P(o), provided that one or more voltage sensors are activated.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel

Activation of BK(Ca) channels by direct Ca(2+) binding and membrane depolarization occur via independent and additive molecular processes. The "calcium bowl" domain is critically involved in Ca(2+)-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca(2+) ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., (912)ELVNDTNVQFLD(923)). To examine these predictions directly, alanine-substituted BK(Ca) channel mutants were expressed in HEK 293 cells and the voltage and Ca(2+) dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1-10 microM free Ca(2+), single point mutations (i.e., E912A and D923A) produced rightward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca(2+)-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca(2+)-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10, and 80 mM cytosolic Mg(2+), this double mutation significantly reduced the Ca(2+)-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca(2+) also reduced direct (45)Ca binding to the calcium bowl domain expressed as a bacterial fusion protein. These findings, along with other recent data, are considered in the context of the calcium bowl's high affinity Ca(2+) sensor and the known properties of EF hands.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).     

Intra- and intersubunit cooperativity in activation of BK channels by Ca2+

The activation of BK channels by Ca(2+) is highly cooperative, with small changes in intracellular Ca(2+) concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca(2+). Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca(2+) sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K(+)) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca(2+) sensors, we examine channels with eight different configurations of functional high-affinity Ca(2+) sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca(2+) activation of the channel when there is only one high-affinity Ca(2+) sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca(2+) sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca(2+) sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca(2+) sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca(2+) response for eight possible subunit configurations of the high-affinity Ca(2+) sensors as well as for three additional configurations from a previous study.     

Cysteine modification alters voltage- and Ca(2+)-dependent gating of large conductance (BK) potassium channels

The Ca(2+)-activated K+ (BK) channel alpha-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 I(K) and shifts the G(K)-V relation to more negative voltages, whereas MTSES and NEM shift the G(K)-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 P(o) 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 Ca(2+)-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 G(K)-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.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Direct regulation of BK channels by phosphatidylinositol 4,5-bisphosphate as a novel signaling pathway

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)) only through phospholipase C (PLC)-generated PIP(2) metabolites that target Ca(2+) stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP(2) activates BK channels independently of PIP(2) metabolites. PIP(2) enhances Ca(2+)-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP(2) acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP(2) (diC16). The PIP(2)-channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6-S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP(2)-induced activation is drastically potentiated by accessory beta(1) (but not beta(4)) channel subunits. Moreover, PIP(2) robustly activates BK channels in vascular myocytes, where beta(1) subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP(2) effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+beta(1) and PI4-kinaseIIalpha robustly activates BK channels, suggesting a role for endogenous PIP(2) in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP(2) antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP(2) levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP(2) directly activates vascular myocyte BK channels to control vascular tone.     

Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.     

Modes of operation of the BKCa channel beta2 subunit

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.