Conopeptide Vt3.1 Preferentially Inhibits BK Potassium Channels Containing β4 Subunits via Electrostatic Interactions*

Background: BK channel function is differentially modulated by tissue-specific β (β1–4) subunits. Results: Conopeptide Vt3.1 preferentially inhibits neuronal BK channels containing the β4 subunit. Conclusion: Electrostatic interactions between Vt3.1 and the extracellular loop of β4 decrease voltage-dependent activation of the channel. Significance: Vt3.1 is an excellent tool for studying the structure, function, and roles in neurophysiology of BK channels. BK channel β subunits (β1–β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

BK channel ␤ subunits (␤1-␤4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous ␤ subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the ␤4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and ␤4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the ␤4 subunit, providing structural insight into the molecular interactions between slo1 and ␤4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying ␤ subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.
Large conductance, voltage, and Ca 2ϩ -activated K ϩ (BK) channels regulate neuronal excitability (1,2), neurotransmission (3), and circadian rhythm (4). BK channels are also important for physiological processes other than in the nervous system such as smooth muscle contraction (5). BK channels are formed by a tetramer of slo1 ␣ subunits that contain the voltage sensor, Ca 2ϩ -binding sites, and the pore (6). Four auxiliary ␤ subunits, ␤1-␤4, which are distributed in a tissue-specific manner, modulate functional properties of the channel, thereby providing a major mechanism for tissue-specific phenotypes of BK channels (7)(8)(9). The ␤4 subunit is predominantly expressed in brain where it is more abundant than any other ␤ subunits (9,10). Therefore, the BK channels comprised of slo1 ϩ ␤4 are considered as the neuronal type. Although the association of ␤4 subunits is important for BK channel function, the molecular mechanism of how various ␤ subunits modulate specific characteristics of channel function and the role of these channels in different physiological processes are still not clear.
Marine predatory cone snails produce conotoxins and conopeptides to stun and paralyze animals for prey capturing and defense. The conotoxins and conopeptides modulate the function of ion channels, transporters, and surface receptors in nervous and muscular systems for fast action to enable the slow moving cone snails against their agile opponents (11). Conotoxins or conopeptides are short peptides consisting of 10 -40 amino acid residues. They are classified by the "superfamilies" according to highly conserved signal sequences in the precursors as well as "families" based on different characteristic cysteine arrangements and different targets (12). Generally, there are two or more disulfide bonds in conotoxins, but only one or no disulfide bonds could be found in conopeptides (12). Among venom peptide families -, -, O-, ␦-, -, and -conotoxins have been demonstrated to interact with voltage-gated Ca 2ϩ , Na ϩ , and K ϩ channels (11,13). These peptide toxins have been valuable tools in studying the structure-function relations and physiological roles of ion channels. A number of ion channel targeting conotoxins have also been used to diagnose ion channel-associated diseases and as drug candidates to affect important biological processes (13). Among these conotoxins, the -conotoxin MVIIA targets on the N-type Ca 2ϩ channels that are related to algesia in the nervous system (14). Based on MVIIA, the synthetic peptide named ziconotide became the first cone snail-derived drug approved by the United States Food and Drug Administration in use for the treatment of severe chronic pain (15,16).
Vt3.1 was identified from Conus vitulinus by cDNA cloning using the M superfamily signal sequence (17). However, unlike other M superfamily conotoxins, Vt3.1 represents a novel group of conopeptides, being a disulfide-cross-linked dimer with an unusual amino acid sequence (see Fig. 1A). After in vitro refolding of the Vt3.1 peptide, two distinct fractions, Vt3.1 (with cross-disulfides) and Vt3.2 (with parallel disulfides) could be purified. Mice showed hyperactivities upon 20 g of Vt3.1 by intraventricular injection. On the other hand, Vt3.2 at the same dose of injection did not cause behavioral abnormality in mice (17). We have screened several conopeptides on their modulation of BK channel function and found that Vt3.1 preferentially inhibited the channel comprised of mslo1 ϩ ␤4 by altering voltage-dependent activation via electrostatic interactions with the channel protein. Using Vt3.1 as a unique probe, these studies showed that the extracellular loop of the ␤4 subunit is important for modulating BK channel voltage-dependent gating and revealed structural features of slo1-␤4 interaction.

EXPERIMENTAL PROCEDURES
Chemical Synthesis and in Vitro Refolding of Vt3.1-The linear Vt3.1 peptide and all of its mutants were synthesized chemically, refolded in vitro, and purified as previously described (17).
Electrophysiology-Macroscopic and single channel currents were recorded from outside-out patches formed with borosilicate pipettes with 1.0 -3.5 M⍀ resistance. The data were acquired using an Axopatch 200-B patch-clamp amplifier (Axon Instruments, Union City, CA) and Pulse acquisition software (HEKA Electronik, Lambrecht/Pfalz, Germany). Recordings were digitized at 20-s intervals and low pass filtered at 10 kHz with the 4-pole Bessel filter built in the amplifier. Capacitive transients and leak currents were subtracted using a P/5 protocol. Experiments were performed at room temperature (20 -22°C , and channel open probability was fitted using clampfit 9 (Axon Instruments, Inc., Union City, CA). For macroscopic currents, G-V relations were measured from the tail currents and fitted with the following Boltzmann equation, where G is conductance, V1 ⁄ 2 is the voltage at which the channels are half-activated, and S is the slope factor. Igor Pro (Wave-Metrics, Inc., Lake Oswego, OR) was used for curve fittings.
Toxin Vt3.1 and Vt3.2 were dissolved in the extracellular solution at 1 mM as stock, and aliquots were stored in Ϫ80°C and diluted to the indicated concentrations (see Figs. 1-6) before experiment. The dose-response curve in Fig. 2B was fitted to the following equation, where I/I 0 is the ratio of current after and before Vt3.1 application, A is the fraction of the remaining current at saturating amounts of Vt3.1, [T] is Vt3.1 concentration, and K is the concentration of Vt3.1 for half-maximum inhibition.
Model of Vt3.1-The structure of a monomeric Vt3.1 peptide was generated by using PEP-FOLD, a de novo structure prediction server (21), which was then optimized by long time scale molecular dynamics simulations using the NAMD program (version 2.9) (22).
Specifically, the CHARMM 27 all-atom force field (49) was used. During each simulation, the protein was solvated in a cubic periodic box of TIP3P water molecules that extended 10 Å from the protein. Each simulation consisted of two preparation steps. First, the system was minimized by 10,000 steps of energy minimization. Second, the system was slowly heated up from 0 to 298 K over a period of 0.5 ns with a harmonic constraint of 1 kcal·mol Ϫ1 · Å Ϫ2 placed on all backbone atoms. Then, non-constrained MD stimulations were performed at constant pressure (1 atm) and constant temperature (298 K) for 80 ns. The SHAKE algorithm (50) was applied for bond constraints, and the particle mesh Ewald (PME) method (51) was used to calculate electrostatic interactions. A cutoff of 1.2 nm was used for the Lennard-Jones interactions. For each simulated system, the trajectory snapshots were saved every 2.0 ps; a total of 40,000 conformations were collected for further analysis.

Vt3.1 Preferentially Inhibits BK Channels Containing the ␤4
Subunit-During the screen of peptide conotoxins against BK channels, we found that Vt3.1 (Fig. 1A) inhibits channels formed by the mslo1 subunit; the maximal conductance in the presence of toxin is smaller than that of control ( Fig. 1, B and C).
To examine whether any ␤ subunit can alter the toxin inhibition, we measured the effect of 10 M Vt3.1 on the currents from coexpression of mslo1 with the ␤1, ␤2, ␤3b, and ␤4 subunit, respectively. The toxin inhibition was slightly reduced by the coexpression of the ␤1 subunit, but not altered by the ␤2 or ␤3b subunits; on the other hand, the ␤4 subunit enhanced the inhibition by more than 2-fold (Figs. 1, C and D, and 2A). The toxin inhibition showed a weak voltage dependence. At Ϫ120 mV, the tail current corresponding to the maximal conductance elicited by a ϩ80 mV test pulse was reduced by 58.8 Ϯ 4.8% for mslo1 ϩ ␤4 (Fig. 1, B and D), whereas the current at the ϩ80 mV test pulse per se was reduced by 34.3 Ϯ 2.8% (Fig. 1, B and C). Similar to mslo1, the mslo1 ϩ ␤4 channels respond to Vt3.1 quickly, with a fast inhibition time constant of 6.4 Ϯ 1.7 s and then followed with a slower current decrease of a time constant Ն20.2 Ϯ 5.7 s. The effect of Vt3.1 can be readily washed out, with a time constant of 7.4 Ϯ 0.6 s ( Fig. 2A). Vt3.1 did not completely inhibit the currents even with the concentration ([Vt3.1]) up to 100 M. The inhibition of the mslo1 ϩ ␤4 by [Vt3.1] can be fitted by a dose-response curve with the maximal inhibition of 71% and the [Vt3.1] at half-inhibition (IC 50 ) of 8.5 M (Fig. 2B).
Vt3.1 Inhibits Voltage-dependent Gating of BK Channels-The results in Fig. 1D and 2B suggest that the toxin may not inhibit BK channels by directly blocking the pore, but instead by affecting the gating mechanism of the channel. In the presence of Vt3.1, the G-V relation is shifted to more positive voltages (also see Fig. 3C), whereas the maximum conductance is reduced (Fig. 1D). However, even at the saturation of Vt3.1 binding, the channel activity is not completely inhibited, allow- Testing voltage pulses were Ϫ200 to ϩ120 mV with ϩ20 mV increments. Holding potential was Ϫ80 mV; repolarization potential was Ϫ80 (mslo1) or Ϫ120 mV (mslo1 ϩ ␤1, mslo1 ϩ ␤4).   FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8 ing the pass of ionic currents (Fig. 2B). Consistent with this mechanism, in the presence of Vt3.1, the channels that remained open showed an altered voltage-dependent activation, with a slower activation and a faster deactivation time course (Fig. 3A). The changes in activation and deactivation kinetics developed in time alongside the onset and washout of the Vt3.1 inhibition of currents (Fig. 3B). Normalized conductance-voltage (G-V) relations of the channel shifted to more positive voltages by 45.5 mV in the presence of 10 M Vt3.1 (IC 50 ϭ ϳ8.5 M; Fig. 2B) (Fig. 3C). All these results indicate that the toxin alters voltage-dependent transitions among the kinetic states during voltage-dependent activation such that the forward activation rate is reduced, whereas the backward deactivation rate accelerated to result in a lower steady state open probability.  (Fig. 3G). Taken together, these results suggest that Vt3.1 inhibits BK channels by altering voltage-dependent gating mechanisms.

Vt3.1 Inhibits BK Potassium Channels with ␤4 Subunit
Vt3.1 Inhibits BK Channels via Electrostatic Interactions with Slo1 and ␤4-The ␤ subunits of BK channels share a common membrane topology, with two membrane-spanning segments, short cytoplasmic N and C termini and a long extracellular loop (Fig. 4A). To investigate the molecular basis for the ability of the ␤4 subunit to enhance Vt3.1 inhibition, we made chimeras between the ␤1 and ␤4 subunits by switching the extracellular loop and then coexpressed each with mslo1. Vt3.1 inhibition of the channel was enhanced as long as the coexpressed ␤ subunit contained the extracellular loop of ␤4 (Fig. 4A), indicating that the amino acids in the extracellular loop of ␤4 that are not conserved in ␤1 are important for the channel protein to interact with Vt3.1. Sequence alignment of the extracellular loop in ␤ subunits reveals the residues that are conserved among ␤1, ␤2, and ␤3b but different in ␤4. We mutated each of these residues in ␤4 to the corresponding residues in ␤1 but found that these mutations did not alter the ability of the ␤4 subunit to

Vt3.1 Inhibits BK Potassium Channels with ␤4 Subunit
enhance Vt3.1 inhibition (Fig. 4B, Group 1 mutations). This finding indicates that these residues are not critical in the interaction with Vt3.1. Next we mutated each of the negatively charged residues in ␤4 that are not conserved among ␤1, ␤2, or ␤3b (Fig. 4B, Group 2 mutations) to examine possible interactions of these residues with the positive charges on Vt3.1 (Fig.  1A). Coexpression of these ␤ mutants with mslo1 showed that a number of the mutations reduced the ability of the ␤4 subunit to enhance Vt3.1 inhibition, resulting in a phenotype more similar to that of the ␤1 subunit (Fig. 4B). These mutations did not alter the effects of the ␤4 subunit on BK channel gating. For instance, while reducing the Vt3.1 inhibition by the largest extent, the E64Q mutation did not change the G-V relations of mslo1 ϩ ␤4 (Fig. 4C), whereas the E64Q ␤4 prolonged the time course of BK channel activation similarly to the wt ␤4 (Fig. 4D) (9). These results suggest that the mutation of the negatively charged residues specifically disrupted the interaction between the channel protein and Vt3.1.
The results in Fig. 4 suggest that the negatively charged residues in the ␤4 subunit extracellular loop enhance the interaction between the channel protein and Vt3.1, leading to an increased Vt3.1 inhibition. To examine whether Vt3.1 also interacts with the negatively charged amino acids in mslo1 channels, we neutralized each of the acidic residues in the extracellular domain of mslo1 by mutation. Glutamic acid 11 is located in the extracellular N terminus of mslo1, and the mutation E11Q eliminated the response of mslo1 to Vt3.1 such that the maximal conductance or the G-V relation changed little in the presence of 10 M Vt3.1 (Fig. 5, A and B). Neutralization of other negatively charged residues also reduced Vt3.1 inhibition of the mslo1 channel (Fig. 5B), suggesting that Vt3.1 interacts with negatively charged residues in the BK channel protein in either the presence or the absence of the ␤4 subunit to inhibit voltage-dependent activation of the channel. The coexpression E11Q mslo1 ϩ ␤4 could be inhibited by Vt3.1 (Fig. 5, C and D), indicating that the interaction of Vt3.1 with ␤4 is not affected by the disruption of the interaction of Vt3.1 with mslo1. These results suggest that the ␤4 subunit provides additional interactions with Vt3.1 through negatively charged residues in the extracellular loop to enhance the toxin inhibition of the channel; these negatively charged residues are located throughout the extracellular loop of the ␤4 subunit and may be close to the negative charges in the extracellular loops of slo1 such that the charges in both slo1 and ␤4 can interact with Vt3.1, suggesting a large interaction surface between the slo1 subunit of BK channels and the ␤4 subunit at the extracellular domains (see below).
Structure of Vt3.1 Is Important for Its Inhibition of BK Channels-Vt3.1 is a dimer of two identical Vt3.1 peptides connected via two cross-formed disulfide bonds, i.e., the disulfide bonds formed between Cys 9 of one peptide and Cys 11 of the other peptide (Fig. 1A). The same two peptides can form a different transient dimer, Vt3.2, via two parallel disulfide bonds, i.e., the disulfide bond formed between Cys 9 of each peptide and the disulfide bond formed between Cys 11 of each peptide (Fig.  6A) (17). Unlike Vt3.1, Vt3.2 at 10 M was unable to inhibit mslo1 ϩ ␤4 (Fig. 6, A and B). To examine whether the two pairs of charged residues in Vt3.1, Arg 4 and Arg 5 , are critical for the interaction between Vt3.1 and the channel protein, we mutated each to Ala. The mutant toxin could no longer inhibit the channel (Fig. 6C). However, we later found that Ala mutation of most of other toxin residues also abolished the ability of Vt3.1 to inhibit the channel (Fig. 6C). These results suggest that Vt3.1 may adopt a conformation that is required for its ability to inhibit mslo1 ϩ ␤4; the different disulfide bond formation in Vt3.2 or mutations in the toxin peptide may disrupt the conformation, leading to the loss of toxin function.  , V1 ⁄2 is the voltage where G-V relation is at half-maximum) for WT and mutant mslo1 channels. N, N terminus; S1-S2: the linker between the S1 and S2 transmembrane segment; S5-P, the linker between the S5 transmembrane segment and the pore helix. * and **, see Fig.  4 (n ϭ 12). C, normalized I-V of E11Q mslo1 ϩ ␤4 without (open circles) and with (filled circles) 10 M Vt3.1 (n ϭ 6). D, the effects of Vt3.1 on current amplitude at ϩ100 mV for WT and E11Q mslo1 ϩ ␤4.  FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 4739
To understand how Vt3.1 inhibits the channel, we modeled the structure of Vt3.1 (Fig. 7) using structure prediction tools and molecular dynamics simulations. Vt3.1 lacks ordered secondary structures (Fig. 7A), which is consistent with the previous circular dichroism analysis (17). Each single chain in the Vt3.1 dimer presents an "L" conformation, where Cys 9 of one peptide and Cys 11 of the other peptide form cross-intermolecular disulfides. The two flexible N termini exhibit positive electric potentials and are close to each other, forming a positively charged and flexible domain (Fig. 7B). A perspective of the structures of Vt3.1 and the Kv1.2 K ϩ channel (23) is shown (Fig.  7C). Comparing to Kv1.2, the slo1 subunit of BK channels has an additional S0 transmembrane segment (24); the positions of transmembrane segments of mslo1 and ␤4 were mapped based on disulfide cross-linking studies (25) (Fig. 7D). The extracellular loop of the BK channel ␤ subunits extends to the central pore based on previous biophysical and pharmacological studies (26,27), but it is not known how the loop relates to other parts of slo1. Our results suggest that the acidic residues in the ␤4 extracellular loop may be located close to the N terminus, the S1-S2 and S5-P linkers, because they all interact with the positive charges in Vt3. 1 (Figs. 4,5,and 7D). Taken together, our results suggest that the interactions between Vt3.1 and the mslo1/␤4 channel may alter voltage sensor movements or the coupling between voltage sensors and the pore to reduce voltage-dependent activation of the channel.

DISCUSSION
Vt3.1 belongs to a novel class of conopeptides (17). Our results show that Vt3.1 inhibits BK channels preferentially in the presence of the ␤4 subunit (Figs. 1, 2, and 4), which is the most abundant BK channel ␤ subunit in brain (9,10). Overall, Vt3.1 inhibits the currents by a maximum of 71% at ϩ80 mV, where the G-V is saturated (Fig. 3C) and shifts the G-V relation by 45 mV at approximately half-saturation concentrations. Therefore, the toxin has two effects on channel gating: to decrease the maximum open probability and to shift the G-V relation to more positive voltages (Fig. 1D), and both effects result in a reduction of the current. At physiological conditions, the peak of neuronal action potentials has a voltage of approximately ϩ30 mV, where the two effects of the toxin will accumulatively decrease the BK current amplitude by more than 90%. These results indicate that the toxin has a clear and strong effect on channel gating and can have a significant effect on neuronal excitability. A previous study showed that injecting Vt3.1 into mouse brain induced hyperactive behavior (17). The mechanism of this behavioral change by Vt3.1 is not known, but the effects of Vt3.1 on BK channels suggest that an inhibition of BK channels may play a role. Vt3.1 inhibits the channel via electrostatic interactions among the basic residues in Vt3.1 and the acidic residues in mslo1 and the ␤4 subunit (Figs. 4,  5, and 7).
Previous studies have identified peptide toxins that modulate BK channels from venoms of various animals including scorpion (28 -30), cone snail (31), snake (32), bee (33), and spider (34). Among these toxins, charybdotoxin (ChTx) and iberiotoxin (IbTx) have been intensively studied for molecular pharmacology and used as an effective tool to identify BK channels in various tissues that contributed greatly in the understanding D, model of Vt3.1 interaction with mslo1 ϩ ␤4, viewed from the extracellular side into the membrane. Transmembrane segments S0 -S6 and the pore helix P for four mslo1 subunits (each color represents one subunit), transmembrane segments T1 and T2 as well as the extracellular loop (black lines) of four ␤4 subunits, and one Vt3.1 molecule (thick blue line) are shown. The red circles represent acidic residues in the extracellular loop of ␤4 subunits that are important for Vt3.1 inhibition of the channel (Fig. 4).

Vt3.1 Inhibits BK Potassium Channels with ␤4 Subunit
of physiological processes and the roles of BK channels. ChTx and IbTx bind to the extracellular vestibule of slo1 and block the ionic flux through the channel (35,36). Their binding to the channel is altered by the association of ␤ subunits; ␤1 enhances the binding affinity of ChTx (37) but reduces the sensitivity of the current to IbTx (38), whereas ␤2 and ␤4 reduce sensitivity to ChTx and IbTx inhibition (8,26). Experimental evidence suggests that these effects are mediated by the extracellular loop of the ␤ subunits that extends to the extracellular vestibule of the pore (26,37,39). Similar to ChTx and IbTx, ␤4 reduced inhibition by slotoxin (40). Martentoxin, on the other hand, showed a more complex behavior such that it enhanced currents of BK channels in association with the ␤1 or ␤4 subunit in the presence of high (20 M) intracellular Ca 2ϩ currents while inhibiting the channel with the ␤4 subunit in the presence of low (10 -500 nM) Ca 2ϩ (41,42). Because ␤4 is the major BK channel ␤ subunit in brain, these properties render the existing toxins ineffective as a tool in studying BK channels in neuroscience. On the other hand, Vt3.1 preferentially inhibits BK channels with the association of the ␤4 subunit. In addition, Vt3.1 acts on BK channels fast, and the effects can be readily washed out (Fig. 2); it is formed by small peptides and easy to synthesize. All these characteristics make Vt3.1 an ideal tool uniquely suited in neuroscience involving BK channels.
Unlike previously studied BK channel inhibitory peptide toxins, Vt3.1 modulates voltage-dependent gating instead of blocking the pore of BK channels (Fig. 3). By studying the mechanism of Vt3.1 inhibition of mslo1 ϩ ␤4 channels, we show that Vt3.1 can be used as an effective tool to probe the structure and function of these channels. Multiple acidic amino acids in the extracellular linkers between membrane spanning helices in slo1 (Fig. 5) and the extracellular loop of ␤4 (Fig. 4) contribute to the inhibitory effects of Vt3.1, suggesting a large interaction interface between the slo1 and ␤4, as well as a large interaction interface between Vt3.1 and the channel. Interestingly, the structural model of Vt3.1 (Fig. 7, A and B) fits nicely to the structural model of mslo1 ϩ ␤4 (Fig. 7, C and D), with the positive charges in Vt3.1 tracing acidic residues in the channel protein. This mode of interaction is consistent with the functional data (Fig. 3), and together these results suggest that Vt3.1 inhibits voltage sensor movements or the coupling between the voltage sensor and the pore, or both, to inhibit voltage-dependent activation. It has been shown that the association of the ␤1, ␤2, and ␤4 subunit alters voltage sensor movements of slo1 (43), and the extracellular loop of the ␤1 subunit contributes to such modulations (44). The alteration of the ␤4 subunit extracellular loop by Vt3.1 alters voltage-dependent activation, indicating that the extracellular loop also contributes to the ␤4 modulation of mslo1 voltage-dependent gating mechanism.
BK channels shorten action potentials and contribute to the fast after hyperpolarization in neurons (1), thereby regulating neuronal firing frequency (2) and synaptic transmission (3). Aberrant BK channel function may lead to neurological diseases. A mutation in human slo1 that enhances BK channel Ca 2ϩ sensitivity has been associated with epilepsy and paroxysmal dyskinesia (45,46). Consistently, knockout of the BK channel ␤4 subunit, which enhanced BK channel activity, resulted in seizure in mouse (47). Recently, it has been shown that the fragile X mental retardation protein (FMRP) regulates presynaptic activity by interacting with the ␤4 subunit and modulating BK channel function (48). Therefore, drugs that modulate neuronal BK channels may provide therapy for BK channel-associated neurological diseases. Our results demonstrate that BK channel with ␤4 subunits can be specifically targeted by a compound ( Fig. 1) that interacts with the acidic residues in the extracellular loop of ␤4 (Fig. 4) to modulate channel function.