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.     

Structural Basis for Calcium and Magnesium Regulation of a Large Conductance Calcium-activated Potassium Channel with β1 Subunits

Large conductance Ca²⁺- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary β subunits, play important roles in diverse physiological activities. The β1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca²⁺ sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and β1 remains elusive. Using macroscopic ionic current recordings in various Ca²⁺ and Mg²⁺ concentrations, we identified two binding sites on the cytosolic N terminus of β1, namely the electrostatic enhancing site (mSlo1(K392,R393)-β1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-β1(L5,V6,M7)), passing the physical force from the Ca²⁺ bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg²⁺ sensitivity. A comprehensive structural model of the BK(mSlo1 α-β1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.     

Interaction sites between the Slo1 pore and the NH2 terminus of the beta2 subunit, probed with a three-residue sensor

Calcium- and voltage-gated (BK) K(+) channels encoded by Slo1 play an essential role in nervous systems. Although it shares many common features with voltage-dependent K(V) channels, the BK channel exhibits differences in gating and inactivation. Using a mutant in which FWI replaces three residues (FIW) in the NH(2) terminus of wild-type beta2-subunits, in conjunction with alanine-scanning mutagenesis of the Slo1 S6 segment, we identify that the NH(2) terminus of beta2-subunits interacts with the residues near the cytosolic superficial mouth of BK channels during inactivation. The cytosolic blockers did not share the sites with NH(2) terminus of beta2-subunits. A novel blocking-inactivating scheme was proposed to account for the observed non-competition inactivation. Our results also suggest that the residue Ile-323 plays a dual role in interacting with the NH(2) terminus of beta2-subunits and modulating the gating of BK channels.     

Structural basis for toxin resistance of beta4-associated calcium-activated potassium (BK) channels

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.     

Gating properties conferred on BK channels by the beta3b auxiliary subunit in the absence of its NH(2)- and COOH termini

Both beta1 and beta2 auxiliary subunits of the BK-type K(+) channel family profoundly regulate the apparent Ca(2)+ sensitivity of BK-type Ca(2)+-activated K(+) channels. Each produces a pronounced leftward shift in the voltage of half-activation (V(0.5)) at a given Ca(2)+ concentration, particularly at Ca(2)+ above 1 microM. In contrast, the rapidly inactivating beta3b auxiliary produces a leftward shift in activation at Ca(2)+ below 1 microM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583-605, this issue), we have shown that some of the apparent beta3b-mediated shift in activation at low Ca(2)+ arises from rapid unblocking of inactivated channels, unlike the actions of the beta1 and beta2 subunits. Here, we compare effects of the beta3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the beta3b subunit and compare it to beta3b constructs lacking either the NH(2)- or COOH terminus or both. The results demonstrate that, although the NH(2) terminus appears to be the primary determinant of the beta3b-mediated shift in V(0.5) at low Ca(2)+, removal of the NH(2) terminus reveals two other interesting aspects of the action of the beta3b subunit. First, the conductance-voltage curves for activation of channels containing the beta3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the beta3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between beta and alpha subunits can affect BK channel function. The COOH terminus of the beta3b subunit produces no discernible functional effects.     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.     

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.     

Four-turn alpha-helical segment prevents surface expression of the auxiliary hbeta2 subunit of BK-type channel

Large conductance, voltage- and Ca2+-activated K+ (BK) channels encoded by the mslo alpha and beta2 subunits exist abundantly in rat chromaffin cells, pancreatic beta cells, and DRG neurons. The extracellular loop of hbeta2 acting as the channel regulator influences the rectification and toxin sensitivity of BK channels, and the inactivation domain at its N terminus induces rapid inactivation. However, the regulatory mechanism, especially the trafficking mechanism of hbeta2, is still unknown. With the help of immunofluorescence and patch clamp techniques, we determine that the hbeta2 subunit alone resides in the endoplasmic reticulum, suggesting that trafficking mechanism of hbeta2 differs from that of hbeta1 opposite to what we predicted previously. We further demonstrate that a four-turn alpha helical segment at the N terminus of hbeta2 prevents the surface expression of hbeta2, that is, the helical segment itself is a retention signal. Using the c-Myc epitope-tagged extracellular loop of hbeta2, we reveal that the most accessible site by antibody is located at the middle of the extracellular loop, which might provide clues to understand how the auxiliary beta subunits regulates the toxin sensitivity and the rectification of BK-type channels.     

NMR structure of the "ball-and-chain" domain of KCNMB2, the beta 2-subunit of large conductance Ca2+- and voltage-activated potassium channels

The auxiliary beta-subunit KCNMB2 (beta(2)) endows the non-inactivating large conductance Ca(2+)- and voltage-dependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BKbeta(2)N) using NMR spectroscopy and patch clamp recordings. BKbeta(2)N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK alpha-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BKbeta(2)N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20-45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BKbeta(2)N-mediated inactivation.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Competitive and synergistic interactions of G protein beta(2) and Ca(2+) channel beta(1b) subunits with Ca(v)2.1 channels, revealed by mammalian two-hybrid and fluorescence resonance energy transfer measurements

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel.     

Lysine-rich extracellular rings formed by hbeta2 subunits confer the outward rectification of BK channels

The auxiliary beta subunits of large-conductance Ca(2+)-activated K(+) (BK) channels greatly contribute to the diversity of BK (mSlo1 alpha) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hbeta2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hbeta2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K(+) concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K(+) inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hbeta2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary beta subunits.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

Voltage and calcium use the same molecular determinants to inactivate calcium channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.     

Differential effects of beta 1 and beta 2 subunits on BK channel activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.     

How Does the W434F Mutation Block Current in Shaker Potassium Channels?

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH₂-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10⁻⁵ or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.     

Species-specific Differences among KCNMB3 BK beta3 auxiliary subunits: some beta3 N-terminal variants may be primate-specific subunits

The KCNMB3 gene encodes one of a family of four auxiliary beta subunits found in the mammalian genome that associate with Slo1 alpha subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct beta3 subunits, beta3a-d. Three variants, beta3a-c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse beta3 variants. Whereas beta1, beta2, and beta4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse beta3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, beta3a and beta3b, of the four found in humans. Both human and mouse beta3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hbeta3b exhibits rapid inactivation, the putative mbeta3b does not inactivate. Furthermore, unlike hbeta3, the mbeta3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca(2+) concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mbeta3b N terminus may not be a true orthologue of the hbeta3b N terminus and that both beta3c and beta3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous beta3 subunits may differ among mammalian species; second, the specific physiological roles of homologous beta3 subunits may differ among mammalian species; and, third, some beta3 variants may be primate-specific ion channel subunits.     

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a “shutter-like” mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.     

Modulation of the BK channel by estrogens: examination at single channel level

BK channels regulate vascular tone by hyperpolarizing smooth muscle in response to fluctuating calcium concentrations. Oestrogen has been reported to lower blood pressure by increasing BK channel open probability through direct binding to the regulatory beta1-subunit(s) associated with the channel. The present investigation demonstrates that 17beta-oestradiol activates the BK channel complex by increasing the burst duration of channel openings. A subconductance state was observed in 25% of recordings following the addition of 17beta-oestradiol and could reflect uncoupling between the pore forming alpha1-subunit and the regulatory beta1-subunit. We also present evidence that more than one beta1-subunit is required to facilitate binding of 17beta-oestradiol to the channel complex.