Zn2+ Activates Large Conductance Ca2+-activated K+ Channel via an Intracellular Domain*

Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn2+ functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn2+ signaling are not well known. We show here that intracellular Zn2+ potently and reversibly activates large-conductance voltage- and Ca2+-activated Slo1 K+ (BK) channels. The full effect of Zn2+ requires His365 in the RCK1 (regulator of conductance for K+) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp367 and Glu399, also reduced activation of the channel by Zn2+, suggesting a possible structural arrangement for Zn2+ binding by the aforementioned residues. Extracellular Zn2+ activated Slo1 BK channels when coexpressed with Zn2+-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn2+ signaling and may participate in sculpting cellular response to an increase in intracellular Zn2+ concentration.

Zinc is the second most abundant transition metal in the human body, playing a pivotal role in the normal development and growth. The utmost importance of zinc is evidenced by the diverse array of symptoms that could result from a chronic dietary deficiency of zinc (1). Biochemically, zinc serves as an essential structural and a catalytic component in many metalloproteins (2), in which the metal is typically coordinated by four or five ligands (3). Multiple zinc coordination geometries are known, but histidine and cysteine typically act as essential ligands (4).
In addition to its role as an integral structural and catalytic factor, Zn 2ϩ is increasingly recognized as a potential intracel-lular signaling molecule, similar to Ca 2ϩ (5,6). Like intracellular Ca 2ϩ , intracellular Zn 2ϩ is normally kept to a very low concentration, from pM to nM (5). Although measurements of free intracellular Zn 2ϩ concentrations ([Zn 2ϩ ] i ) in living cells remain challenging, studies do suggest that [Zn 2ϩ ] i may significantly increase under some conditions. For example, a robust release of Zn 2ϩ from the endoplasmic reticulum, termed "zinc wave," has been observed in response to extracellular stimuli, further suggesting that Zn 2ϩ may act as an intracellular second messenger (7). In addition, local [Zn 2ϩ ] i may be significantly higher near Zn 2ϩ -permeable channels (5,8), analogous to the well known micro-and nano-domains of intracellular Ca 2ϩ (9). Moreover, [Zn 2ϩ ] i may increase concomitantly with [Ca 2ϩ ] i under pathological conditions such as ischemia/hypoxia (5,6,10,11), in which intracellular Ca 2ϩ overload is suspected to contribute to cell death in these conditions (12). However, whether such increases in [Zn 2ϩ ] i contribute to the deleterious effect or play a compensatory cell-protective effect is not clear (5,11,(13)(14)(15)(16).
Large-conductance voltage-and Ca 2ϩ -activated K ϩ (BK Ca , Slo1 BK or K Ca 1.1) channels are distinguished by their allosteric activation by voltage and intracellular Ca 2ϩ (17)(18)(19). Like other voltage-gated K ϩ channels, a BK channel complex includes four pore-forming ␣ (Slo1) subunits, each of which contains a voltage sensor domain (S1-S4) and one-fourth of the ion conduction pore (S5-S6) (20). In addition, each Slo1 subunit possesses the transmembrane segment S0 (21) and a large cytoplasmic area harboring two homologous domains termed "regulators of conductance of potassium" (RCK1 and RCK2) essential for activation by Ca 2ϩ for the channel (22,23). Functionally, BK channels participate in many crucial physiological phenomena including vasoregulation, synaptic transmission, and hormone secretion mainly by affecting membrane excitability (17). In addition, as a feedback controller of intracellular Ca 2ϩ , BK channel activation has been demonstrated to have a potent cell protection effect by limiting the influx of Ca 2ϩ during hypoxia/ischemia (24,25).
The concomitant increases in [Zn 2ϩ ] i and [Ca 2ϩ ] i in ischemia/hypoxia and the cytoprotective role of the BK channel under the pathological conditions prompted us to examine whether Zn 2ϩ is also a physiological activator of the channel. The Slo1 protein indeed contains multiple putative Zn 2ϩ -binding amino acid sequences such as HXXXH (X represents any amino acid) identified in other metal-binding proteins including S100 proteins, the largest subgroup of the EF-hand Ca 2ϩbinding protein family (26 -28). Our excised patch clamp measurements from heterologously expressed human Slo1 (hSlo1) 3 BK channels revealed that intracellular Zn 2ϩ robustly activates the channel and that mutation of one histidine residue in the RCK1 domain fully abolished the stimulatory effect of Zn 2ϩ . Our results therefore suggest that Slo1 coordinates Zn 2ϩ using amino acid ligands in the RCK1 domain and that the Slo1 BK channel is a positive effector of intracellular Zn 2ϩ signaling.
Electrophysiology and Data Analysis-Ionic currents were recorded using the cell-attached or excised inside-out configuration at room temperature. Patch electrodes (Warner) had a typical initial resistance of 1.5-2.0 megohms. The series resistance, up to 90% of the initial input resistance, was electronically compensated in the macroscopic current measurements. Macroscopic capacitive and leak currents were subtracted using a P/6 protocol. The current signal was filtered at 10 kHz through the built-in filter of the patch clamp amplifier (AxoPatch 200A; MDS Analytical Technologies) and digitized at 100 kHz using an ITC-16 AD/DA interface (HEKA). Conductance-voltage (G-V) curves were generated from tail currents and fitted with a Boltzmann equation as described (29). The resulting half-activation voltage (V 0.5 ) was used to quantify the effect of Zn 2ϩ on the channel. Both activation and deactivation time constants were obtained by fitting the currents with a single exponential excluding the initial 180 s. The results were analyzed as described using IGOR Pro (WaveMetrics) (29). Statistical comparisons between two groups were performed using the unpaired or paired t test, as appropriate. Comparison of more than two groups was performed using analysis of variance followed by a Tukey HSD test as implemented in IGOR Pro. Statistical significance was assumed at p Յ 0.05, and the data are presented as mean Ϯ S.E. The number of samples in each group is shown in parentheses unless noted otherwise.
Chemicals and Solutions-All chemicals were from Sigma except for 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA; Biotium). TPEN was dissolved in dimethyl sulfoxide and diluted with the internal recording solution to the final concentration of 10 M. The final concentration of dimethyl sulfoxide (0.02%, v/v) did not affect Slo1 channel currents. For inside-out patch recording, the extracellular solution contained 140 mM KCl, 2 mM MgCl 2 , 10 mM HEPES, pH 7.2, with N-methyl-D-glucamine (NMDG

RESULTS
Intracellular Zn 2ϩ Activates hSlo1 Channels-To observe the effect of cytoplasmic Zn 2ϩ on the Slo1 channel while maintaining a very low concentration of Ca 2ϩ , we used KF in the internal solution in which most of the contaminating Ca 2ϩ precipitated due to the low solubility of CaF 2 . In such an internal solution, the free Ca 2ϩ concentration has been estimated to be Ͻ20 nM (30). Consistently, we found that the activity of the hSlo1 channel remained unaltered when the inside-out patches were transferred from the KF internal solution (see "Experimental Procedures") to the KCl internal solution with 11 mM EGTA in which [Ca 2ϩ ] is calculated to be Ͻ10 nM (WEBMAXC STANDARD; data not shown). In addition, we found that up to 300 M of Zn 2ϩ in the KF solution failed to activate the smallconductance Ca 2ϩ -activated channel 2 (SK2), which has higher Ca 2ϩ sensitivity than the Slo1 channel (9) (data not shown). These observations together affirmed that [Ca 2ϩ ] i was appropriately buffered to a negligible level when Zn 2ϩ was added into the KF internal solution.
Addition of Zn 2ϩ (0.3-300 M) quickly and reversibly increased hSlo1 BK currents (Fig. 1, A and C) in a concentrationdependent manner (Fig. 1B). TPEN, a Zn 2ϩ chelator with low affinity for Ca 2ϩ , fully antagonized the stimulatory effect of the Zn 2ϩ addition to the intracellular solution ( Fig. 1, C and D), further confirming that it was Zn 2ϩ that increased the hSlo1 current. In contrast, extracellular Zn 2ϩ , up to 2 mM, was without any stimulatory effect (see Fig. 6B; see also Ref. 31).
The current-enhancing effect of Zn 2ϩ was voltage-dependent (Fig. 1E) and accompanied by a shift in G-V to the hyperpolarized direction without any change in the steepness (Fig. 1F). Saturating concentrations of Zn 2ϩ (Ն100 M) produced a shift in V 0.5 of about Ϫ75 mV. The Zn 2ϩ -dependent shift in G-V V 0.5 had an EC 50 value of 33.6 Ϯ 12.2 M and a Hill coefficient of 0.93 Ϯ 0.22 (Fig. 1G).
We noticed that high concentrations of Zn 2ϩ slightly diminished the peak outward currents at extreme positive voltages (e.g. 200 mV in Fig. 1E) without decreasing the inward tail current size. This small inhibitory effect, most probably reflecting voltage-dependent block of the channel pore by Zn 2ϩ (30), was not investigated any further. In addition to the shift of voltage dependence of activation to the hyperpolarized direction, Zn 2ϩ slowed the deactivation kinetics without affecting the activation kinetics (Fig. 1, H and I).
The stimulatory effect of Zn 2ϩ was also observed at the single-channel level. Zn 2ϩ drastically increased single-channel open probability in a wide range of voltages, including a physiologically relevant negative voltage (Ϫ50 mV) and an extreme negative voltage where the primary voltage sensors of the channel are not activated (Fig. 1J). Zn 2ϩ had no noticeable effect on the unitary current size (Fig. 1J).
Zn 2ϩ -dependent Activation of the hSlo1 BK Channel Did Not Require the Conserved Zinc-binding Motifs-Structural studies suggest that histidine and cysteine are the two most frequently used zinc ligands in metalloproteins, in which zinc interacts with the imidazole nitrogen or thiol sulfur in the conserved zinc-binding motifs such as HXXXH, and CXXXH (X represents any amino acid) (2-4, 27, 28, 32). Mutation of either histidine residue in the conserved motif typically disrupts the Zn 2ϩ coordination and reduces catalytic activity of metalloenzymes (26,33). Inspection of the hSlo1 sequence shows that the cytoplasmic domain of the channel contains three putative zinc-binding motifs, 464 HNKAH 468 , 749 HELKH 753 , and 612 CKACH 616 , localized in RCK1, RCK2, and the linker region between the two RCK domains, respectively ( Fig. 2A). To assess the contributions of His and Cys to the Zn 2ϩ -induced Slo1 BK channel activation, we utilized diethyl pyrocarbonate, a histidine-modifying reagent (34), and a cysteine-modifying reagent, MTSEA (35). Our results showed that pretreatment of the channel with diethyl pyrocarbonate significantly attenuated the Zn 2ϩ -induced activation of the channel, decreasing the V 0.5 shift to ϳ50% of that observed in the control group (Fig. 2, B and H). In contrast, MTSEA failed to alter the Zn 2ϩ -induced channel activation (Fig. 2, C and H). We thus reasoned that the Slo1 protein interacts with Zn 2ϩ using histidine residues, possibly in the aforementioned zinc motifs ( Fig. 2A).
The potential involvement of the histidine residues in the zinc-binding motifs in the channel was further tested by mutation of His 464 , His 616 , His 749 , and His 753 . A robust stimulatory effect of Zn 2ϩ , indistinguishable from that in the wild-type channel, remained in these His-to-Arg mutants (Fig. 2, D-F). The mutant channel H616R (29) did not express well enough to record macroscopic currents; however, the mutant retained a Zn 2ϩ sensitivity indistinguishable from that of the wild-type channel based on single-channel measurements (Fig. 2G). These results collectively indicated that the His residue(s) that coordinate Zn 2ϩ are located elsewhere.
Zn 2ϩ Is Less Effective at Low pH-The bound Zn 2ϩ can be removed from the metalloenzymes in low pH conditions, possibly owing to the protonation of imidazole nitrogens (36). We therefore examined whether intracellular H ϩ affected the action of Zn 2ϩ on the Slo1 channel. The Zn 2ϩ -induced shift in V 0.5 was indeed significantly reduced in the pH 6.2 internal solution to Ϫ25.1 Ϯ 7.1 mV, less than a half of that at pH 7.2 (p Ͻ 0.01; Fig. 3, A and F).
Mutation of His 365 Abolishes the Zn 2ϩ Effect-We previously demonstrated that two His residues, His 365 and His 394 , in the RCK1 domain serve as the primary H ϩ sensors of the hSlo1 channel and mediate pH-dependent activation of the channel (37,38). The antagonistic effect of low pH on the Zn 2ϩ -dependent activation suggests that the same His residues may be required for the Zn 2ϩ action. Consistent with this possibility, the double mutation H365R/H394R completely abolished the effect of Zn 2ϩ on V 0.5 ; the ⌬V 0.5 value was Ϫ2.8 Ϯ 5.1 mV (p Ͻ 0.001 compared with the wild-type channel; Fig. 3, B and F). Of the two His residues, His 365 clearly plays the most important role, for the single mutation H365R alone eliminated the Zn 2ϩ sensitivity (⌬V 0.5 ϭ Ϫ5.5 Ϯ 2.9 mV; p Ͻ 0.0001 compared with the wild-type channel; Fig. 3, C and F). Mutation of His 365 to neutral alanine (H365A) also completely disrupted the Zn 2ϩ sensitivity of the channel (Ϫ6.0 Ϯ 3.9 mV; p Ͻ 0.001 compared with the wild-type channel and p Ͼ 0.5 compared with H365R) (Fig. 3, D and F). In contrast, the mutant H394R remained fully Zn 2ϩ -sensitive (⌬V 0.5 ϭ Ϫ55.2 Ϯ 1.2 mV; p Ͼ 0.5; Fig. 3, E and F). While both His 365 and His 394 in the RCK1 domain are important for pH-dependent activation of the hSlo1 channel (37,38), only His 365 is required for the Zn 2ϩ -dependent activation of the channel.
Select Acidic Residues in the RCK1 Domain Implicated in the Ca 2ϩ Sensitivity Are also Important for the Zn 2ϩ Action-His 365 , required for the Zn 2ϩ -dependent activation of the hSlo1 channel (see Fig. 3) also participates in both Ca 2ϩ -and H ϩ -dependent activation of the Slo1 channel such that the stimulatory effect of H ϩ is diminished at higher concentrations of Ca 2ϩ (37,38). We hypothesized that Ca 2ϩ may also interfere with the Zn 2ϩ -dependent activation of the channel. As predicted by this idea, we found that in the presence of 100 M Ca 2ϩ , which is a saturating concentration for the high-affinity Ca 2ϩ sensors of the Slo1 channel (39 -42), Zn 2ϩ failed to alter G-V (Fig. 4,  B and F), indicative of a functional competition between Zn 2ϩ and Ca 2ϩ . Previous mutagenesis studies suggest the presence of at least three potential divalent cation sensors in each Slo1 subunit (18,(41)(42)(43) (Fig. 4A); a high-affinity sensor in the RCK1 domain, a high-affinity Ca 2ϩ bowl sensor, and a low affinity sensor in the RCK1 domain that also mediates Mg 2ϩdependent activation of the channel (42). The chargeneutralization mutation D367A in the RCK domain is known to disrupt the high-affinity Ca 2ϩ -sensing by the RCK1 domain (41). We found that the mutation significantly decreased the shift in V 0.5 by 100 M Zn 2ϩ by ϳ35% to Ϫ36.9 Ϯ 5.6 mV (p Ͻ 0.01 compared with the wild-type channel; Fig. 4, C and F). The function of the high-affinity Ca 2ϩ bowl sensor in the RCK2 domain is disrupted by the deletion mutation ⌬884 -885 (39). This deletion mutation, however, failed to alter the stimulatory effect of Zn 2ϩ on the channel (Fig. 4, D and F).
The low-affinity divalent cation sensitivity of the Slo1 channel is in part mediated by Glu 399 in the RCK1 domain (43). The mutation E399A, which impairs the stimulatory action of mM levels of Mg 2ϩ on the channel (42,43), noticeably attenuated the Zn 2ϩ -dependent shift in V 0.5 by ϳ35% to Ϫ37.2 Ϯ 1.8 mV  (p Ͻ 0.01 compared with the wild-type channel; Fig. 4, E and F). The shifts in V 0.5 by Zn 2ϩ in the D367A and E399A mutants were statistically indistinguishable (Fig. 4F).
Other transition metals, such as Mn 2ϩ , also activate the Slo1 channel (30,42). We found that the effect of Mn 2ϩ was completely disrupted by the mutation E399A but not by the mutation H365A, which eliminates the Zn 2ϩ sensitivity (supplemental Fig. S1).
Coexpression of ␤1 Subunit Does Not Alter the Effect of Zn 2ϩ -In addition to the four pore-forming Slo1 subunits, a native BK channel complex may also include auxiliary ␤ subunits in a tissue-dependent manner (44). Heterologous coexpression of the auxiliary subunit ␤1, predominantly expressed in the cardiovascular system, dramatically increases the overall Ca 2ϩ sensitivity and slows both the activation and deactivation kinetics of the channel complex (44). The underlying mechanism is postulated to involve an increase in the Ca 2ϩ affinity of the high-affinity Ca 2ϩ sensors in the RCK1 domain and the Ca 2ϩ bowl in the RCK2 domain (45). Because the stimulatory effect of Zn 2ϩ on the Slo1 channel was in part dependent on Asp 367 , an established component in the high-affinity RCK1 Ca 2ϩ sensor (41), we examined whether coexpression of ␤1 enhanced the effectiveness of Zn 2ϩ . Functional coexpression of ␤1 was verified by the characteristically slower activation and deactivation kinetics. We found that Zn 2ϩ remained effective in enhancing the Slo1 current. The shift in V 0.5 (Ϫ56.8 Ϯ 2.4 mV) was indistinguishable from that without coexpression of ␤1 ( Fig. 5; p Ͼ 0.5).
Extracellular Zn 2ϩ Activates Slo1 BK Channel when Coexpressed with TRPM7-Many membrane transport proteins including ion channels mediate translocation of the extracellular Zn 2ϩ into intracellular space. Extracellular Zn 2ϩ did not affect the Slo1 channel activity; however, it robustly activated the channels when they were coexpressed with TRPM7, a non-  selective cation channel permeable to Zn 2ϩ (46,47). In contrast, extracellular Mg 2ϩ did not alter Slo1 channel open probability (Fig. 6).

DISCUSSION
Zn 2ϩ is well known for its structural role in a large number of metalloproteins, including some voltage-gated K ϩ channels in which the metal ion mediates tetramerization of the channel proteins (48,49). As an important intracellular messenger, Zn 2ϩ also modulates multiple signaling pathways, but yet only a small number of its direct effectors have been clearly identified (5,7). Among ion channels, recent studies show that the TRPA1 (transient receptor potential channel A1) (50,51) and the ATPsensitive K ϩ channel (K ATP ) (52) are activated by intracellular Zn 2ϩ at nM and M concentrations, respectively. Our study now adds the Slo1 channel as a new member of the Zn 2ϩ signaling cascades. Heterologously expressed Slo1 BK channels are robustly activated by M levels of intracellular Zn 2ϩ in cell-free membrane patches, independently of the auxiliary subunit ␤1. Moreover, mutation of His 365 in the RCK1 domain or nearby Asp 367 or Glu 399 involved in the Ca 2ϩ sensing fully or partially abolished the channel activation by Zn 2ϩ .
Our finding that Zn 2ϩ activates heterologously expressed Slo1 channels is in contrast with a previous report that Zn 2ϩ had no effect on rat skeletal muscle BK channels incorporated in planar lipid bilayers (30). The reason for the apparent discrepancy is not clear. It may be noted that the authors also failed to observe any stimulatory effect of Mg 2ϩ , an established activator of Slo1 channels (43,53) in the same study (30).
The Mechanism of Channel Activation by Zn 2ϩ -The functional competition between Zn 2ϩ and Ca 2ϩ in activation of the Slo1 channel observed in this study is in line with the mutagenesis result that Asp 367 , essential for the normal high-affinity Ca 2ϩ sensing of the channel (41), is also required for Zn 2ϩ action. Accordingly, the mechanism of channel activation by Zn 2ϩ may be similar to that by Ca 2ϩ . Although physical measurements of Ca 2ϩ binding to the RCK1 sensor and the Ca 2ϩ bowl sensor are preliminary (54 -56), conformational changes in an isolated hSlo1 cytoplasmic domain induced by Ca 2ϩ have been detected (54). Structural and functional studies of MthK and Slo1 suggest that Ca 2ϩ -dependent activation of the Slo1 channel may be accompanied by an expansion of the cytoplasmic domain termed a "gating ring" (22), the mechanical energy of which is further coupled to the channel pore (19,57). Like Ca 2ϩ , Zn 2ϩ may induce a similar expansion of the gating ring to promote activation of the channel. However, some differences between the effects of Ca 2ϩ and Zn 2ϩ exist. The maximal shift in V 0.5 by Zn 2ϩ , about Ϫ75 mV, is clearly smaller than that by Ca 2ϩ , which can produce a shift of Ϫ200 mV at 300 M (42). One readily discernible reason for the difference is that the Ca 2ϩ action is supported by both the sensor in the RCK1 domain and the Ca 2ϩ bowl sensor in the RCK2 domain (39,41,54,58). Even in the absence of the Ca 2ϩ bowl sensor, 300 M Ca 2ϩ can produce a Ϫ125 mV shift (41), still greater than that by Zn 2ϩ . The maximal V 0.5 shift by Zn 2ϩ is similar to that caused by H ϩ , which also works via the RCK1 sensor and functionally competes with Ca 2ϩ (37). The smaller shift by H ϩ is attributed to weaker allosteric coupling between the gate of the channel and the RCK1 sensor when H ϩ is bound as compared with that with Ca 2ϩ bound (38). Thus, the coupling strength in the presence of Zn 2ϩ may be similarly lower than that with Ca 2ϩ . Another difference between the effects of Ca 2ϩ and Zn 2ϩ relates to Glu 399 in the RCK1 domain, a critical component in the low-affinity divalent ion sensing of the channel, and its neutralization impairs the channel activation by mM levels of Mg 2ϩ (43). Whereas the stimulatory effect of M levels of Ca 2ϩ does not depend on Glu 399 , the effect of Zn 2ϩ is diminished when Glu 399 is neutralized (Fig. 4). The action of Zn 2ϩ is thus influenced by the residues involved in both the high-affinity and low affinity divalent cation sensing mechanism (19). The biophysical mechanism of the channel activation by Zn 2ϩ may be similar to that by Ca 2ϩ because, unlike effect of Mg 2ϩ (59), the Zn 2ϩ action remains effective even at negative voltages where the voltage sensors of the channel are not activated. Finally, coexpression with ␤1 enhances the shift in V 0.5 by Ca 2ϩ but does not alter that by Zn 2ϩ (Fig. 5). A similar ␤1-indepenent effect is observed with intracellular H ϩ (37,38) further supporting the idea that Zn 2ϩ and H ϩ may share a similar mechanism in Slo1 channel activation.
Zinc Coordination by Slo1-In many metalloproteins that contain zinc as a stable cofactor, the metal is coordinated by a water molecule and three to four ligands provided by the amino acid residues, typically the side chains of His, Glu, Asp, and Cys (4). Some proteins coordinate zinc using His, Asp, and Glu (4). In Slo1, at least His 365 , Asp 367 , and Glu 399 contribute to the stimulatory effect of Zn 2ϩ and His365 is required. The lack of a high-resolution atomic structure of the channel, however, precludes a detailed inference on the zinc coordination geometry. Furthermore, unlike most other zinc-containing proteins, binding of Zn 2ϩ to the channel is rapid and readily reversible, and it is not clear how applicable the structural information obtained from the metalloproteins that contain zinc as a stable cofactor may be to the Slo1 protein. Many intracellular EFhand Ca 2ϩ -binding proteins also reversibly bind to Zn 2ϩ at concentrations similar to those used to activate Slo1 BK channels (28). Structural studies suggest that Zn 2ϩ is often located in close proximity to Ca 2ϩ sites and that the two ions reciprocally modulate binding of the other (60,61), in agreement with our finding that Ca 2ϩ and Zn 2ϩ competitively activate Slo1 BK channel. The RCK1 domain, which contains the His residue essential for the Zn 2ϩ action, was once postulated to contain an EF-hand-like domain (55). However, subsequent structural studies on the prokaryotic channel MthK, which shares a high level of sequence similarity in this area with the Slo1 BK channel, did not support this idea (22,23). The homology model of Slo1 (Fig. 4A) (62) based on the MthK structure clearly shows that Asp 367 and Glu 399 , are located in the vicinity of His 365 , forming a potential ligand binding pocket that accommodates a Ca 2ϩ , H ϩ , or carbon monoxide (37,63). The requirement for His and the contributions from Asp and Glu in Zn 2ϩ activation of the Slo1 channel are in line with the zinc coordination schemes found in metalloproteins such as an Escherichia coli rhamnose isomerase (4,64). We therefore suggest that His 365 , Asp 367 , and Glu 399 in the RCK1 sensor coordinate Zn 2ϩ , and the conformational change of the sensor promotes opening of the gate. In TRPA1 channels, which are also activated by intracellular Zn 2ϩ , His and Cys residues located some distance away in the primary sequence appear to play a critical role in the Zn 2ϩ sensitivity (51).
Physiological and Pathophysiological Implications-Our study demonstrated that human Slo1 BK channels were activated by high nM to M of intracellular Zn 2ϩ . Similar concentrations were also used in Zn 2ϩ modulation of other intracellular proteins such as mitochondrial enzymes (65)(66)(67) and ion channels (52). For instance, the EC 50 for activation of recombinant K ATP channels, sulfonylurea receptor (SUR)1/Kir6.2 and SUR2A/Kir6.2 are 1.8 and 60 M, respectively (52). Such [Zn 2ϩ ] i may not be observed physiologically in the bulk intracellular compartment. However, local [Zn 2ϩ ] i may reach higher levels near intracellular Zn 2ϩ stores or Zn 2ϩ permeable channels and it plays important roles in normal neuronal transmission and immune response (5,7,68). Interestingly, some Zn 2ϩ -permeable ion channels may physically colocalize with Slo1 BK channels, potentially exposing the latter to a locally high level of Zn 2ϩ (69). Although quantitative studies of such local Zn 2ϩ domains are unavailable, functional analyses of local Ca 2ϩ domains suggest that the [Ca 2ϩ ] i near Slo1 BK channels can be a few orders of magnitude greater than the mean bulk concentration (9,69). Thus it is plausible that the local [Zn 2ϩ ] i increases transiently to a M level to activate Slo1 BK channels. Our results (Fig. 6) show that such an increase in [Zn 2ϩ ] i could occur through an influx of Zn 2ϩ from the extracellular compartments mediated by Zn 2ϩ -permeable TRPM7 channels (46,47,70). The extracellular concentration of Zn 2ϩ in confined compartments such as synaptic clefts may reach several hundred M (5). Because both TRPM7 and Slo1 BK channels are widely expressed, TRPM7 channels could inject enough Zn 2ϩ to activate Slo1 BK channels. Along with TRPA1 (50, 51) and K ATP channels (52), Slo1 BK channels now represent a family of intracellular Zn 2ϩ -activated ion channels that could play physiological roles. Increases in [Zn 2ϩ ] i may be even greater under some pathological conditions such as brain ischemia/reperfusion and epilepsy (5,12). For example, in the experimental seizures induced by kainic acid, [Zn 2ϩ ] i may increase to hundreds of nM and several M in hippocampal and cortical neurons, respectively (71,72). A recent study suggests that the actual increase in [Zn 2ϩ ] i during brain ischemia and reperfusion may be significantly more than previously estimated because the divalent cation overload traditionally thought to be from Ca 2ϩ , is actually from Zn 2ϩ (10). This interpretation and the observation that [Ca 2ϩ ] i may reach 30 M during ischemia (12) together indicate the actual [Zn 2ϩ ] i may be in the M range, sufficient to activate Slo1 BK channels, suggesting that Zn 2ϩdependent activation of Slo1 BK channels may play a role during cerebral ischemia. The finding that pharmacological activation of BK channels is cell protective during ischemic stroke (24,25) indicates that the Zn 2ϩ -dependent activation of the channel probably represents a compensatory and adaptive response.
In summary, this study demonstrates that hSlo1 BK channels are intracellular Zn 2ϩ -activated channels and represent a new effector of intracellular Zn 2ϩ signaling. The stimulatory effect of Zn 2ϩ requires His, Asp and Glu in the RCK1 domain. As a member of the Zn 2ϩ -signaling cascade, Slo1 BK channels may participate in many phenomena mediated by intracellular Zn 2ϩ , particularly in some diseases associated with a significant increase in [Zn 2ϩ ] i .