LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 385 Biol Res 39: 385-401, 2006 BR

REVIEW Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

RAMÓN LATORRE1 and SEBASTIAN BRAUCHI1, 2

1 Centro de Estudios Científicos, Valdivia, Chile. 2 Universidad Austral de Chile, Valdivia, Chile.

ABSTRACT

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) 2+ transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca , and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.

Key terms: BK channel, Ca2+-binding sites, voltage dependence, allosteric models.

PROLOGUE: ON SQUIDS AND THE AMAZING scientifically independent. In other words, MONTEMAR LABORATORY all the scientists at Montemar followed the Peter Medawar’s dictum to the young If you ask me (RL) why the laboratory in scientist apprentice: choose an important Montemar was so important in the problem and become apprenticed to a senior development of biophysics in Chile, I would scientist. I fully agree with Perutz when he immediately say: because the seniors were states that “creativity in science, as in the doing good science, and they let the juniors arts, cannot be organized. It arises to do whatever they wanted. In this regard, spontaneously from individual talent”. Well- our professors made it clear from the onset run laboratories, as was the case of of our research work that we were supposed Montemar, can foster talent, but hierarchical to take one idea from the many floating organizations, inflexible bureaucratic rules around in the lab in those years and develop that plague our universities, and mountains the experimental and theoretical framework of futile paperwork can kill it, as is usually in an absolutely independent manner. the case in Chile. Freedom is a terrible thing when one is The next thing I learned during my Ph.D. young and insecure, and learning to be on years at Montemar was that biology is above my own was the first thing I was forced to all an experimental field and that no theory master in Montemar. Our teachers were wise is as valuable as a well-done experiment. enough and brave enough to give us Actually, many experiments were needed, sufficient intellectual ammo to make us and the squid season was short, so you were

Corresponding author: Ramón Latorre, Centro de Estudios Científicos, Arturo Prat 514, Valdivia, Chile, E-mail: [email protected] Received: July 22, 2005. Accepted: August 22, 2005 386 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 forced to work hard and efficiently and process, you can use this knowledge to your even built your own equipment when advantage in order to overcome poverty. necessary (voltage clamps were not In Montemar, I realized that science is commercially available at the time!). The not a quiet life. If you put together two whole lab environment forced you to realize Italian descendants (Mario and Mitzy) in that “experimentation is a form of thinking the same space, you get an explosive and that a wrong interpretation of an mixture. Discussions about science, lab experiment is forgivable, but an space, or politics were frequently heated unrepeatable experimental result is not” and always won by Fernando Vargas (Medawar, 1979). because during the course of a debate he Paul Ehrlich, the father of immunology, never lost his temper, and his logic was used to say that scientists need the four Gs: unbeatable. So, there was another lesson for Geschick, Geduld, Geld, und Glück (skill, us: scientists are generally passionate patience, money, and luck). Science at people who defend their territories and pet Montemar was undertaken with ideas as ferociously as bears. Above all, considerable skill, patience and luck but however, our teachers taught us that in very little money; our advantage over the Montemar the authority principle did not rich laboratories of the United States was exist. If you were wrong, somebody was the squid (Dosidicus gigas). Biophysics there to show you that you were mistaken, first became a Chilean specialty because whether you were a graduate student, “that’s where the squids were” as Chris Luxoro, or the Pope himself. One of his Miller stated in an interview that appeared characters in Fred Hoyle’s novel the Black in Science in 1995. Using the squid giant Cloud remarks that scientists are always axon, Mario Luxoro and his pupil Eduardo wrong, yet they always go on. What makes Rojas (“Guayo” to his colleagues and them to continue? I think it is something students) were the first to claim that that was always latent in Montemar: the proteins were involved in the electrical passion for solving problems aesthetically. excitability of nerve (Rojas and Luxoro, Eduardo Rojas deserves a special space 1963). The axons of this monster, about one in these remembrances. Guayo was millimeter in diameter, were, during the fundamental in my life as a scientist. Due to 1960s, an attraction difficult to resist for the political situation in the country during scientists interested in nerve excitability. in the late 1960s, most of our professors left And this is another important reason why Montemar to take different posts at the the Montemar laboratory was so successful. Universidad de Chile, and Cecilia and I Every summer, we were visited by were left orphaned of advisers. During the scientists of the stature of Clay Armstrong, summer of 1969, Guayo adopted us, and Ichigi Tasaki, Bob Taylor, and Gerry thanks to his support (emotional and Ehrenstein. Clay, Gerry, and Guayo were scientific), we were able to finish our Ph.D. my first scientific idols, Clay because of his theses. His generosity and kindness made amazing wit, Gerry because of the economy possible for me to be telling you my part of and precision of his thinking, and Guayo the Montemar adventure. because he was able to play Beethoven with a voltage-clamp set-up. Clay, Gerry and Guayo were the first to introduce me to the INTRODUCTION amazing field of ion channels. Thanks to the squid, Montemar was a window to the When Mario Luxoro asked me (RL) to world, and Cecilia Hidalgo, Pancho write a chapter in this issue of Biological Bezanilla, and I had the opportunity (Glück) Research dedicated to my teacher, of doing postdocs in very good laboratories colleague, and friend, Eduardo Rojas, I in the U.S. Thus, the “little ones” also immediately thought that the best present learned at Montemar that if you have a for him would be to say something about unique biological preparation that enables the ion channel that is closest to my heart. you to understand a fundamental biological From the moment we discovered its LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 387 existence in a membrane preparation from the diversity of BK channels is great. muscle T-tubule (Latorre et al., 1982), we Regulatory β-subunits, splicing, and were confronted with a molecular metabolic regulation create this diversity Pandora’s box: once opened, its electrical fundamental to the adequate function of language left all of us bewitched. Others many tissues (Vergara et al., 1998; Latorre (e.g., Marty, 1981; Pallota et al., 1981) et al., 2000; Orio et al., 2002) In a bird’s- were as fascinated as we were with this eye view of this fantastic molecular “monster” of a single-channel conductance machine, we will discuss its molecular closed to the ceiling imposed by simple properties and how these properties diffusion combined with an exquisite K+ determine the BK channel opening and selectivity. BK channels essentially are closing. impermeant to Na+ and conduct K+ 10- and 200-fold more effectively than Rb+ and Cs+, respectively (large conductance channels GROSS BK CHANNEL STRUCTURE AND THE CA2+- were not supposed to be so selective!) SENSING ELEMENTS (Blatz and Magleby, 1984; Eisenman et al., 1986; Stefani et al., 1997). At the same The cloning of the BK channel from time, the channel was activated by voltage Drosophila (Atkinson et al., 1991; Adelman and cytoplasmic Ca2+. This latter property et al., 1992) showed that the BK channel led Meech (1978) to hypothesize that this was a member of the S4 superfamily conductance system was perfect link encompassing voltage-dependent K+ (Kv), between cell metabolism and electrical Na+ and Ca2+ channels. The gene coding for activity, and he was right on the mark. BK was called Slowpoke or Slo and, after Because of its large conductance, this the cloning and expression of Slo2 (Yuan et voltage and calcium-activated K+ channel al., 2000; Yuan et al., 2003) and Slo3 was christened “maxi K” (Latorre and (Schreiber et al., 1998), was renamed Slo1. Miller, 1983) or “BK” (for big K+; Blatz In the case of Kv channels, the channel- and Magleby, 1987). forming protein possesses six Muscle contraction, neurosecretion, transmembrane domains (S1-S6) containing chromaffin cell electrical activity, and hair the pore-forming domain S5-P-S6 and an cell tuning are some of the key S4 voltage-sensing element. Like Kv physiological processes that require an channels, the BK channel is a tetramer increase in cytoplasmic Ca2+ to develop. (Shen et al., 1994); unlike Kv channels, Most often, this Ca2+ increase is mediated however, the BK channel protein consists by the Ca2+ entry into the cells through of seven transmembrane domains (S0-S6) voltage-dependent Ca2+ channels (VDCCs). that lead to an exoplasmic N-terminus (Fig. The increase in internal Ca2+, however, also 1; Meera et al., 1997; Wallner et al., 1996; puts into action a negative feedback that Toro et al., 1998). The large C-terminus will serve to stop or to dampen excitatory (containing about twice as many amino phenomena induced by the opening of acids as S0-S6) has four hydrophobic VDCCs. This negative feedback appears as domains (S7-S10), and Salkoff’s group a consequence of the activation of BK identified two molecular domains (S0-S8, (about 250 pS in 100 mM symmetrical KCl; “core” and S9-S10, “tail”) that, when Marty, 1981; Pallota et al., 1981; Latorre et expressed together, were able to produce al., 1982; for reviews see Latorre et al., functional channels (Wei et al., 1994). 1989; McManus, 1991). Thus, BK channels have the largest single-channel conductance of all K+ selective channels. To ensure SEARCHING FOR THE HIGH AFFINITY CA2+- maximum efficiency of the negative BINDING SITES feedback, BK channels functionally co- localize with VDCCs (Marrion and Tavalin, Taking advantage of the fact that the 1998; Prakriya and Lingle, 1999). Despite Drosophila Slo (dSlo1) channel has a being coded by a single gene (Slowpoke), different Ca2+ sensitivity than the mouse 388 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401

Figure 1. Schematic diagram of the α (black) and β (gray) subunit of BK channels. A. D362/D367, M513 and the calcium bowl define sites that when mutated decrease the high- affinity Ca2+ sensitivity of the BK channel. E374/E377 is a site that when mutated decreases the low affinity Ca2+ and Mg2+ sensitivity. The first two sites are located in the RCK domain. The RCK domain is defined as the amino acid stretch comprised between the C-terminus of S6 and the C- terminus of S7. B. BK channels are tetramers. C. Primary sequence of the Ca2+ bowl aligned with the corresponding partial sequence of the C-terminus in Slo3 and the mutant D5N5. LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 389

Slo (mSlo1) channel, Wei et al. (1994) bowl and that, in the absence of Ca2+, the showed that coexpression of mSlo core Slo1 tail inhibits voltage-dependent BK together with dSlo tail produced channels channel gating, inhibition that is relieved by with a Ca2+ sensitivity similar to that of the Ca2+. This inhibition may help to keep the dSlo channel. These experiments provided channel closed in the resting cell (low the first indication that the tail was playing internal Ca2+ and negative membrane the role of the calcium-sensing element. potential). A detailed single-channel Closer inspection of the tail showed the analysis confirmed the results of Schreiber presence of a domain consisting of 28 et al. (1999) and suggested that in addition amino acids containing nine acidic residues to playing a role as a Ca2+ sensing domain, including a string of five conserved the tail domain also modulates the gating aspartates residues, the “calcium bowl” and conductance properties of BK channels (Fig. 1A, C). Partial deletion or point (Moss and Magleby, 2001). mutations of the aspartates contained in the In all the experiments described above, calcium bowl produced BK channel that Ca2+-binding sites were inferred from the were less sensitive to Ca2+ - at the same effects of Ca2+ on channel activation (i.e., Ca2+ concentration, the calcium bowl changes in the BK open probability). Bian mutant conductance-voltage (G-V) curves et al. (2001) measured direct binding of were right-shifted compared to the wild- radioactive calcium to a COOH-terminus type BK channel G-V curve (Schreiber and fragment of the Drosophila BK containing Salkoff, 1997; Bian et al., 2001; Braun and the calcium bowl and showed that mutating Sy, 2001). If the Ca2+ bowl is disrupted by the aspartates to asparagines (D5N5 mutant; deleting crucial aspartates, this high Ca2+ Fig. 1C) was able to reduce the Ca2+- affinity regulatory site is lost, but the binding affinity by about 60%. This result mutant and the wild-type BK showed the demonstrates a direct correlation between same sensitivity to Cd2+ ions. This finding Ca2+ binding and Ca2+ sensitivity for provided evidence of the presence of two channel activation (see also Braun and Sy, different Ca2+-binding sites; a site sensitive 2001). The D5N5 mutant expressed in cells to Ca2+ but not to Cd2+ and another site able exhibits a lower Ca2+ sensitivity for to bind with similar affinity Ca2+ and Cd2+ activation and reduces the dSlo channel’s (Schreiber and Salkoff, 1997). Clearly, it Hill coefficient 2-fold, as if one binding site would be difficult to explain the per monomer is lost in the D5N5 mutant. observation that BK activation is sensitive The most economic way to account for to a range of Ca2+ concentrations that spans these results is to assume the existence of over four orders of magnitude on the basis two distinct Ca2+-binding sites per channel of a single Ca2+-binding site (see Fig. 6). monomer (Bian et al., 2001; cf. Schreiber The Slo3 channel lacks the calcium bowl and Salkoff, 1997). Bao et al. (2004) (Fig. 1C) and is not sensitive to Ca2+. Co- explored the relative importance of the expression of mSlo1 core and mSlo3 tail different acidic amino acids contained in gave origin to Ca2+-insensitive channels in the calcium bowl using alanine scanning the range comprised between 0-10 μM mutagenesis of 20 residues contained in this internal Ca2+, but the data presented domain. These experiments were done in indicated that the chimeric Slo channels the background mutant, M513I; a mutant were activated by Ca2+ at concentrations that eliminates one of the high ≥300 μM (Schreiber et al., 1999). Ca2+sensitivity regulatory sites (see below) Interestingly, mSlo1 core-mSlo3 tail allowing determining the isolated Ca2+- channels opened at much lower voltages binding properties of the calcium bowl. than mSlo1 at zero internal Ca2+ They found that mutation to alanines of two (conductance-voltage curves for the critical aspartates (D898 and D900; Fig. chimeric channel were shifted by about 60 1C) has the greatest effect on the change in mV to the left along the voltage axis). the G(V) curve half voltage (ΔV0.5) upon These results suggest the presence of a addition of Ca2+. The point mutations also Ca2+-binding site different from the Ca2+ were able to decrease binding of 45Ca by 390 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 about 50% to a peptide composed of a channel, and the mutant D362A/D367A- fusion protein consisting of glutathione-S- 5D5N is still gated by voltage, with a transferase and a 207-amino acid part of the voltage dependence similar to that of the mSlo1 tail that includes the calcium bowl. wild type, but Ca2+ is unable to activate the These results indicate that there are “hot” channel at [Ca2+] < 10-3 M (Xia et al., residues in the calcium bowl and gave 2002). Another mutation, M513I, a residue further support to the hypothesis that this immediately following the S7 domain (Figs. domain is the one of the high affinity sites 2B and 3B), together with a deletion mutant that couples Ca2+ binding to channel that eliminates most of the aspartates opening. (1) contained in the calcium bowl (Δ896-903), The molecular origin of the remaining similar to the D362A/D367A-5D5N mutant, BK Ca2+ sensitivity after neutralization of was shown to completely remove high most of the acidic residues present in the affinity responsiveness to Ca2+ (Bao et al., Ca2+ bowl was elucidated by mutating 2002). The behavior of the M513I and negatively charged residues outside the Δ896-903 mutants was analyzed in terms of Ca2+ bowl and, in particular, in the allosteric models (Cox and Aldrich, 2000; regulator of conductance for K+ (RCK; Rothberg and Magleby, 2000; Horrigan and Jiang et al., 2001; Fig. 1-3). The RCK Aldrich, 2002; see below) that allow for the domain in the BK channel was unveiled by quantitative determination of the MacKinnon’s group (Jiang et al., 2001) by contribution of the different mutations to multiple sequence alignment of the BK the BK channel activation. The main channel with prokaryotic K+ channels and finding of this study was that in the absence other proteins known to possess the RCK of an applied voltage, each regulatory site domain structure. The structure of the RCK contributes almost equally to the free domain of a six transmembrane domain K+ energy difference between open and closed channel from E. coli (Fig. 2A) solved at 2.4 states. The estimated dissociation constants Å resolution has a Rossmann-fold topology, were in the micromolar range. Thus, a very common structural motif found in although no direct evidence supports the enzymes and ligand-binding proteins. idea that D362/D367 or M513 form part of Rossmann-fold secondary structures are a structure able to coordinate Ca2+ ions, the organized into two linked β−α−β−α−β data of Xia et al. (2002) and Bao et al. units (see Figs. 2B and 3A) and were first (2002) suggest that the BK channel identified in a number of NAD+-dependent contains at least two high affinity Ca2+- dehydrogenases (Darby and Creighton, binding sites. The results of Schreiber and 1993). Salkoff (1997) pointed towards the The hypothesis that BK channels contain existence of a second site able to bind Cd2+ a RCK domain was put to test by disturbing and gave a clear indication of the existence a well-conserved salt bridge, not commonly of a second Ca2+-binding site with different present among Rossmann-fold proteins divalent cation selectivity. Oberhauser et al. (K448-D481 in hSlo1; Fig. 3B). If either (1988), on the other hand, found that the position is mutated (e.g., K448D or BK channel can be activated by a series of D481K), the channel becomes less Ca2+ divalent cations including Sr2+, Cd2+, Mn2+, sensitive. However, the double mutant Fe2+, and Co2+. Taking advantage of these K448D/D481K recovers the Ca-sensitivity observations, Zeng et al. (2005) performed shown by the wild-type BK channel. a series of elegant divalent cation Therefore, these results strongly suggest the selectivity experiments using the D362A/ presence of a salt bridge as predicted from D367A mutant to investigate the selectivity the E.coli K+ channel RCK structure and of the Ca2+ bowl or the D5N5 mutant to support the hypothesis that the BK channel determine the divalent cations able to contains an RCK domain on its C-terminus. activate the channel by interacting with the The double mSlo1 RCK mutant D362A/ D362/D367 regulatory mechanism. The D367A (Fig. 3C) produced a marked results indicated that the calcium bowl was reduction in the Ca2+ sensibility of the BK able to selectively bind Ca2+ and Sr2+, while LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 391

Figure 2. mSlo1 RCK secondary structure prediction. A. Sequence alignment used for the homology modeling. mSlo1 C-terminus primary sequence used was directly taken from the protein data bank (sequence number NP_034740). Available KCH_ECOLI-RCK domain coordinates (P31069, 1ID1) obtained previously by X-ray crystallography (Jiang et al., 2001) were used as template. The alignment used for the homology modeling was performed using LIALIGN. There is ~20% identity between these proteins, but they are similar regarding their secondary structure. The homology modeling was performed using Modeler 7v7 (Marti-Renom et al., 2000). LOOPS routine was used for the unaligned sequences. The models that show the lowest energy profiles were selected. WATH IF web interface was used to check the obtained structures and remove bumps. B. The secondary structure prediction of the hSlo1 RCK domain was performed using JPred (Professor Barton group, Dundee University, Scotland, UK) and PredictProtein Server (Columbia University Bioinformatics Center) (Karplus, 2003; Rost, 2003). Secondary structure prediction was used to evaluate the structural similarity between RCK-Domain from E.coli 6TM channel (KCH_ECOLI) and mSlo1 RCK. Previous alignment between these two sequences was carried out by Jiang et al. (2001). Secondary structure prediction is marked as E (Strand) and H (Helix). The order of Rossmann structures β−α−β is predicted for mSlo1 from βA through αD, after this point, the a helices from Jiang et al. (2001) original structure are conserved but there are no more predicted beta formations. Moreover, original βE in Jiang et al. (2001) structure was replaced by a α helix in our prediction, named αX (highlighted). Buried (B) amino acids also are predicted and indicate that the hydrophobic segment S7 is buried almost completely. Important charges involved in calcium sensitivity are highlighted with red asterisks. Residues forming salt bridges are marked with dotted lines. Notice that E374 is predicted to form a salt bridge with H350. Solid line over the conserved sequence IMRVI, shows a sequence involved in the interaction between domains. 392 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401

Figure 3. mSlo1 RCK-Domain. Different views of the obtained structure are shown. A. General view for the obtained structure. The original β−α−β motif is conserved, and the protein is highly packed. Predictions for buried amino acids are in agreement with the structure in which α−helices form a shelter for β strands. It is possible to distinguish two domains in the structure, the first one is comprised by βA through αD and the second one is defined by what we called αX through αG. S7 hydrophobic segment corresponds to αG and is predicted to be buried. It is possible that this structure is in close contact with the next not-modeled portion of carboxyl terminal. B. Several important residues were highlighted. Yellow sticks are the amino acids involved in the interaction with the other subunit. The interaction between K448 and D481 is conserved, as there is a similar pair present in the E.coli RCK. Residue M513 is shown in a CPK representation (yellow). C. Details of the domain involved in calcium sensitivity. Amino acids D362, D367, E374, and E399 are showed. The amino acids indicated as responsible for Ca2+ low affinity E374 and E399 are accompanied by histamines (H344/H350). (D) A complete view of all the salt bridges formed in the structure. These salt bridges may play a key role in maintaining the structure of the RCK domain. Molecular modeling protocols were performed using Hyper hem 7.5 professional (Hypercube, Inc.) with CHARMM 27 force field. The best model obtained previously was minimized using steepest descent protocol in vacuum in the presence of counter ions. Problematic loops were improved as follows; four simple 20 pHs annealing protocols were done (heating 300k, cooling 100k, cooling steps 1k/0.01ps). Each annealing was followed by 30 cycles of conjugate gradients minimization and 30 cycles of steepest descent minimization or until convergence was reached. Finally, salt bridges were calculated using WHAT IF web interface. Image handling was performed using DS Viewer 5.0 (Acers Inc.) LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 393 regulation defined by D362/D367 can be was discovered that most of these cations activated by Ca2+, Sr2+, and Cd2+. A third, are able to activate the BK channel in the low affinity mechanism (discussed below), absence of Ca2+ by binding to a low affinity can be put into action by Ca2+, Sr2+, Cd2+, site (Shi and Cui, 2001; Zhang et al., 2001; Mn2+, Ni2+, and Co2+. In the absence of Shi et al., 2002; Zeng et al., 2005). binding assays like those performed with Notice in Fig. 4A, that Ca2+ shift the BK carboxyl terminus fusion proteins (Bian probability of opening (Po) vs voltage et al., 2001; Braun and Sy, 2001), there curves towards the left along the voltage exists the possibility, however, that all the axis and that the data is well fitted by using results are due to coincidental allosteric a Boltzmann function of the type: effects on Ca2+ binding to a region that lies outside the RCK region. Independent of 1 these considerations, the unique divalent (1) Po = − zF (V −V ) / RT cation selectivity of the different BK 1+ e 0.5 regulatory mechanisms supports the existence of at least three distinct divalent where z describes the channel voltage cation binding sites and that the sites act dependence, F is Faraday’s constant, V is independently of each other. the applied voltage, and V0.5 , the voltage at Although rooted in solid experimental which Po = 0.5. A convenient way of data, this picture of a BK channel determining the ability of Ca2+ to activate 2+ containing all his Ca -sensing machinery the BK channel is by plotting the V0.5 as a located in the carboxyl terminal, needs function of Ca2+ concentration (Fig. 4B) further confirmation both from binding and because is directly related with the free structural studies, in particular, in view of energy, ΔG to open the channel since the report by Piskorowski and Aldrich

(2002), who claimed that a channel lacking ΔG= zFV05. (2) the calcium bowl and RCK domain can still be activated by Ca2+. However, by where the values of z obtained from the fit of constructing chimeric channels in which the the data shown in Figure 4A to a Boltzmann carboxyl termini between the Slo1 channel function are given in Fig. 4C. This type of (Ca2+ activated) and the Slo3 (pH plot was extensively used in the dependent) were switched, Lingle’s group characterization of BK channels and gave showed that the C-terminus of Slo1 is a the first hint of the existence of a low requisite for channel Ca2+ sensitivity (Xia affinity Ca2+ site (Wei et al., 1994; Cui et al., et al., 2004). The chimeric channel Slo3-C- 1997). Fig. 4B shows that V0.5 shifts to more teminus Slo1 was activated by Ca2+, and the negative voltages as the Ca2+ concentration chimeric channel Slo1-C-terminus Slo3 is increased, but above 10 μM, there is a induced pH-sensitive currents. tendency towards saturation, suggesting that a new binding site has began to play a role in BK channel activation. This low affinity site LOW AFFINITY CA2+-BINDING SITES AND THE is unselective (see above) and Shi and Cui MECHANISM OF MG2+ ACTIVATION OF BK (2001) and Zhang et al. (2001) showed that 2+ Mg was able to produce similar ΔV0.5 in Golowash et al. (1986) found that the presence or in the absence of Ca2+. In millimolar amounts of Mg2+ were able to other words, Mg2+ activates BK channels potentiate BK channel activation by Ca2+ independently of Ca2+. Mg2+ does not affect and to increase the Hill coefficient the activation but slows down the describing the BK Ca2+ activation curves. deactivation kinetics, suggesting that it binds Other divalent cations, such as Cd2+, Mn2+ , preferentially to the open state. The possible Fe2+, Co2+, and Ni2+, also were able to location of the Mg2+ regulatory domain was enhance activation and increase the Hill revealed by co-expressing Slo1 core and coefficient of channels already activated by Slo3 tail. These chimeric channels, which do Ca2+ (Oberhauser et al., 1988). Recently, it not contain the Ca2+ bowl, are still sensitive 394 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 to Mg2+, indicating that the low affinity site of the RCK domain of the BK channel, Shi resides in the RCK domain (Figs. 2 and 3; et al. (2002) concluded that the Mg2+ ion is Shi and Cui, 2001). Sequence alignment of coordinated by the side chains of E374, part of the RCK domain in Slo1 and Slo3 E399, and Q397. Thus, the first RCK revealed several acidic amino acids present domain of the BK channel appears to contain in the Slo1 RCK but not in the Slo3 domain. high and low affinity Ca2+-binding sites. Our These residues that are conserved in mSlo1 own modeling of the RCK domain suggests and dSlo1 but not in Slo3 were mutated to that both E374 and E399 are very close to the corresponding amino acids present in H350 and H344, respectively and that the Slo3 with the result that two hot spots were pair E374/H350 forms a salt bridge (Fig. 3C found, E374A and E399N (Fig. 3C; Shi et and D). It would be interesting to test for the al., 2002). Mutation of either of these two possibility that the low Ca2+ affinity of these residues completely obliterated Mg2+- putative Ca2+ and Mg2+ sites is determined dependent activation. Based on the structure by the close proximity of the histidine proposed by Jiang et al. (2001) for the core residues.

Figure 4. Voltage and Ca2+ dependence of the BK channel. A. Averaged Po(V) curves at the indicated Ca2+ concentrations. Lines are the best fit to a Boltzmann distribution (Esq. 1). Fitted parameters, V0.5 and z, are shown in B and C. Solid line is the best fit to the allosteric model shown in Fig. 6 E. B. V0.5 plotted against Ca2+ concentration. C. z values plotted against Ca2+ concentration. Solid line is the best fit to the allosteric model shown in Fig. 6 E. LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 395

ACTIVATION BY VOLTAGE AND THE VOLTAGE displacement of the charged residues SENSOR contained in the S4-inducing gating currents (Armstrong and Bezanilla, 1973; Despite the fact that it contains an S4 Schneider and Chandler, 1973; Keynes and domain, the molecular nature of the BK Rojas, 1974; Yang and Horn, 1995; channel voltage-dependence remained Aggarwal and MacKinnon, 1996; Seoh et unclear until it was unequivocally al., 1996; Larsson et al., 1996; Yang et al., demonstrated that depolarizing voltages are 1996; Yusaf et al., 1996; Ahern and Horn, able to activate the BK channel in the 2004). Gating currents in BK channels were absence of Ca2+ (Pallota, 1985; Meera et al., detected by Stefani et al., (1997) in the 1996; Cui et al., 1997; Cox et al., 1997; absence of Ca2+ and showed that like the Stefani et al., 1997; Horrigan and Aldrich, G(V) curves, the gating charge-voltage 1999; Rothberg and Magleby, 2000; (Q(V)) curves were left-shifted in the Nimigean and Magleby, 2000; Taludker and presence of Ca2+. The maximum number of Aldrich, 2000). One of the first observations gating charges per channel, obtained by in support of the presence of an integral dividing the limiting charge (Qmax) by the voltage sensor in the BK channel-forming number of channels in the patch, was about protein is depicted in Figure 4B. Figure 4B 4, much less than those obtained in the case clearly shows a region comprised between 5 of the voltage-dependent Shaker K+ channel 2+ and about 100 nM Ca , where the V0.5 is (12-14 electronic charges; Schoppa et al., independent of the Ca2+ concentration 1992; Seoh et al., 1996; Aggarwal and (Meera et al., 1996). Moreover, in this Ca2+ MacKinnon, 1996; Noceti et al., 1996). The concentration interval, the channel can be reason for this large difference in charge maximally activated by voltage, indicating per channel between BK and Shaker is still that in the absence of Ca2+, voltages high unclear, since a primary alignment of hSlo enough can increase the Po to its maximum and Shaker charged domains S2, S3 and S4 value (Cui et al., 1997). shows that most of the negative charges in In voltage-dependent channels, S2 and S3 and positive charges in S4 are membrane depolarization promotes the conserved (Fig. 5).

Figure 5. Alignment of the S2, S3 and S4 transmembrane domains of the human (hSlo) and Shaker K+ channel. Horizontal bars denote the transmembrane segments in both channels. Numbers indicate the position of the far right residues in the primary sequence of their respective proteins. Amino acids in bold are the charged residues conserved in Kv channels and hSlo channels. 396 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401

In Shaker, four arginines (R362, R365, adjacent open and closed intervals: shorter R368, and R371) contribute to the gating closed intervals are preferentially adjacent charge (Seoh et al., 1996; Aggarwal and to longer open intervals and longer closed MacKinnon, 1996) and Bezanilla’s group intervals. Given the large number of closed presented elegant evidence that these four and open states, the correlation between charges move the entire electric field adjacent intervals and the fact that the (Bezanilla, 2000; Starace and Bezanilla, channel is a tetramer, in the absence of 2001; Starace and Bezanilla, 2004). In hSlo, Ca2+, BK channel gating is consistent with three of these arginines are conserved the 10-state model indicated in Fig. 6A. (R207, R210, and R213) but only two of One of the predictions of this model is that them (R210 and R213) were found to be even in the absence of voltage sensor able to alter the maximal slope of the Po(V) activation, described by the equilibrium relationship (Diaz et al., 1998; cf., Cui and constant J, the channel can open through Aldrich, 2000). More recently, Ma and the reaction described by the equilibrium Horrigan (2005) reported that charge D153 constant L. (3) This prediction was in S2 and R213 in S4 reduce the maximum confirmed by Horrigan et al. (1999), who 2+ voltage dependence of Po. (2). If R213 is found that even in the absence of Ca and the only gating charge, it should move all at very negative voltages, the channel can the way across the electric field (one charge open with a very low, but measurable, Po per channel subunit). Considering that in (about 10-6) and a low voltage dependence, Kv channels the voltage sensors structure zL, not related to the voltage sensors. On (S1-S4) are essentially self-contained, the other hand, a detailed analysis of the independent domains inside the membrane gating currents in the absence of Ca2+ (Long et al., 2005a, b) and that BK suggests a two-state model, resting- channels contain an extra transmembrane activated (R-A) suffices to explain the domain (S0), it is probable that the voltage sensor movement (Horrigan and structure of the voltage sensor of the BK Aldrich, 1999). The simple behavior of the channel will prove to be slightly different gating currents, Q(V) curves which are well from that of Kv channels. The clue of the described by a Boltzmann function, the large difference in gating changes between monoexponential kinetics of the fast BK and Kv channels may lie in structural component of the gating current, and the differences in the voltage sensor. lack of a gating current rising phase also are consistent with the kinetic model proposed in Fig. 6A in which the voltage EXPLAINING BK CHANNEL ACTIVITY USING sensors act independently. ALLOSTERIC MODELS In the allosteric model described in Figure 6A, for each voltage sensor In this section, I will emphasize some of the activated, the equilibrium constant for highlights of the two-tiered allosteric model channel opening, L, is multiply by an that is commonly used to explain the BK allosteric factor D, so the opening process channel activity. For details, the reader is facilitated as more voltage sensors are should consult the excellent review by activated. The observation that even when 2+ Magleby (2003). In the absence of Ca , all voltage sensor are resting, Po can be Nimigean and Magleby (2000) and increased by augmenting intracellular Ca2+ Taludker and Aldrich (2000) found 4-5 (Horrigan and Aldrich, 2002) is the basis exponential components in the dwell time for postulating the allosteric kinetic model distribution for the closed states and 2-3 depicted in Figure 6B under the components in the dwell time distribution assumption that there is only one Ca2+- for the open states for the BK channel. binding site per channel subunit. In this Linear models in which only one pathway case, for each Ca2+-binding site occupied led to the open states were excluded since the equilibrium constant L is multiply by McManus et al. (1985) found an inverse an allosteric factor C. Figures 5A and B relationship between the duration of define the key feature of BK channels: LATORRE & BRAUCHI Biol Res 39, 2006, 385-401 397

Figure 6. Allosteric activation model for BK channel. A. The allosteric activation by voltage originates a 10-state Monod-Wyman-Changeaux (MWC) activation model. In this case, the allosteric factor is D and the equilibrium constant J. B. The allosteric activation by Ca2+ also originates a 10-state MWC model. For each Ca2+-binding site occupied, the equilibrium constant for channel opening, L, is multiplied by the allosteric factor C. C. The combination of A and B produces a two-tiered, 50-state model. D. The complete 70-state model takes into account the interaction between the voltage sensor activation and Ca2+ binding (allosteric factor E). When E=1, we recover the 50-state model (modified from Horrigan and Aldrich, 2002; Magleby, 2003). neither Ca2+ nor voltage are strictly The best compromise between simplicity necessary for channel activation and Ca2+ and reproduction of the voltage and calcium binding and voltage sensor activation can dependence in a wide range of voltages and 2+ act independently to enhance channel Ca concentrations, including very low Pos, opening. Thus, we are in the presence of is probably the 50-state two-tiered gating three processes, Ca2+ binding, voltage mechanism shown in Fig. 6C (Rothberg and sensor activation, and channel opening, Magleby, 1999, 2000; Cox and Aldrich, which are independent equilibriums that 2000; Cui and Aldrich, 2000). If some interact allosterically with each other. In allosteric coupling (E; Fig. 6D) between support of the model shown in Fig. 6B, Ca2+ binding and voltage sensor movement Niu and Magleby (2002), using channels is included, the model increases to 70 states with 1, 2, 3 or 4 Ca2+ bowls (4), determined (Horrigan and Aldrich, 2002) and in several that the Hill coefficient increased in a occasions, this has been the model of choice stepwise fashion as the number of bowl (e.g., Orio and Latorre, 2005). The beauty of increased from 1 to 4. This observation is the model is that it is possible to set consistent with models like the one shown experimental conditions to determine some in Fig. 6B in which the Ca2+ binding to of the different parameters unequivocally. each of the sites is independent, and For example, in the absence of Ca2+ and at cooperativity arises as a consequence of the very negative voltages, channel gating action of the allosteric factor C. kinetics is determined by the transition: 398 LATORRE & BRAUCHI Biol Res 39, 2006, 385-401

L Zeng et al., 2005), but evidence for the C↔O second RCK domain is lacking. Niu et al. (2004) studied the effect of and changing the length of the linker joining the S6 with the RCK domain. The authors use a O 1 (3) simple and elegant model in which the Po = = O + C 1+ L−1 coupling could be explained by using a spring-like model. They found that as L << 1, Po ~ L = Loexp(zLFV/RT). Thus, shortening the linker increase channel under these experimental conditions, we are activity and increasing linker length able to determine two parameters: Lo and decreases channel activity. These zL. This exercise allows us to arrive at observations suggest that, in the absence of another important conclusion: for the BK Ca2+, the linker-RCK (gating ring?) channel, the limiting slope is actually complex behaves as a passive spring determined by the lesser voltage-dependent attached to the channel gates. This linker- transition and does not reflect the voltage gating complex in the absence of Ca2+ sensor charge effectively coupled to applies force to the gates, an observation channel activation (see Almers, 1978; Sigg consistent with the finding that compared to and Bezanilla, 1997). The existence of two the Slo3 tail, the Slo1 tail inhibits channel or maybe three Ca2+-binding sites with opening. Ca2+ binding, as in the case of the different affinities (see above) makes the MthK channel, transform the passive spring picture more complicated, however, and into a force generating that uses the free raises almost exponentially the number of energy of Ca2+ binding to pull open the S6 states in a model. helices to permit ion conduction.

SOME INFERENCES ABOUT THE MOLECULAR ACKNOWLEDGEMENTS NATURE OF CA2+ ACTIVATION We thank all the members of the Latorre Jiang et al. (2002) determined the crystal Laboratory for their enthusiasm and much structure of the Ca2+-bound open discussion over the years about the Methanobacterium thermoautotrophicum workings of ion channels. This work was K+ (MthK) channel. The structure shows supported by grants from the Fondo that each of the four channel subunits Nacional de Investigación Científica y contributes with two RCK domains forming Tecnológica (FONDECYT 103-0830 to R. a gating ring where two Ca2+ ions bind per Latorre and FONDECYT 104-0254 to F. subunit. The site behaves as the low Ca2+ González), the Human Frontiers in Science affinity binding site in BK, since millimolar Program. S. Brauchi was recipient of a amounts of Ca2+ are necessary to activate Ph.D. fellowship from the Comisión the MthK channel. Jiang et al. (2002) put Nacional de Ciencia y Tecnología. The forward a model in which Ca2+ binding Centro de Estudios Científicos is a promotes displacement mediated by a Millennium Institute and is funded in part flexible interface of the two RCK domains by a grant from Fundación Andes. expanding the diameter of the gating ring; this expansion is used to mechanically open the pore to an open channel. Whether this NOTES mechanism applies to the BK channel is unclear, but the presence of RCK domains 1. The correlation between the DV0.5 in BK channels is undeniable (Jiang et al., induced by the mutations and the effect on 2001; Figs. 2 and 3). Actually two RCK 45Ca binding was lost when the mutation domains have been proposed to be present D899A was tested. This mutation has no in BK channels, which can act as a gating effect on DV0.5 but a large effect on the ring similar to that of MthK channels (e.g., 45Ca-binding signal. Bao et al. 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