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J. Biol. Chem., Vol. 276, Issue 45, 42116-42121, November 9, 2001

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NMR Structure of the "Ball-and-chain" Domain of KCNMB2, the ₂-Subunit of Large Conductance Ca²⁺- and Voltage-activated Potassium Channels^*,

Detlef Bentrop^§¶, Michael Beyermann, Ralph Wissmann, and Bernd Fakler^§

From the  Department of Physiology II, University of Tübingen, Ob dem Himmelreich 7, 72074 Tübingen, Germany and the  Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin, Germany

Received for publication, July 26, 2001, and in revised form, August 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The auxiliary -subunit KCNMB2 (₂) endows the non-inactivating large conductance Ca²⁺- 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, BK₂N) using NMR spectroscopy and patch clamp recordings. BK₂N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK -subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BK₂N 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 BK₂N-mediated inactivation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Large conductance K⁺ channels (BK¹ or MaxiK channels) are key modulators of excitability in many types of cell (1, 2). They are formed from four identical -subunits encoded by the Slo gene and are activated by membrane depolarization and/or increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i (3-8)). This dual activation is unique among the large family of K⁺ channels and provides a direct feedback mechanism to regulate Ca²⁺ influx.

In many tissues, the activation gating of BK channels is modulated by accessory -subunits, a family of membrane proteins (KCNMB) closely associated with the -subunit (7). Four KCNMB proteins have been identified (KCNMB1-4), and they all share a prototypic topology of two transmembrane domains with intracellular N and C termini (9-13). Functionally, each of these KCNMB proteins distinctly changes the rates of channel activation and deactivation as well as the apparent sensitivity of the channel for Ca²⁺ (9).

In addition, one of the -subunits, KCNMB2 (₂), was found to confer rapid and complete inactivation to the BK channel complex (11, 12) in a manner similar to that observed in chromaffin cells of the adrenal gland or in hippocampal CA1 neurons (14, 15). Analysis of this KCNMB2-mediated inactivation gating showed that it closely resembled the famous ball-and-chain-type inactivation of voltage-gated K⁺ channels (Kv): (i) it is determined by the N terminus of KCNMB2; (ii) it occludes the open channel pore and competes with the pore-blocking agent tetraethylammonium (11, 12); (iii) recovery from inactivation is speeded up by an increase of the extracellular K⁺ concentration (11).

Moreover, the N-terminal stretch of the KCNMB2 N terminus (19 amino acids) was shown to be a functional entity, i.e. its fusion to the N terminus of KCNMB1 (₁) conferred rapid inactivation to this non-inactivating -subunit, and it occluded BK channels as a synthetic peptide very similar to the "pore plugging" observed for the synthetic inactivation domains (ID) derived from various -subunits and one -subunit of Kv-type K⁺ channels (11).

The three-dimensional structure of Kv-derived IDs was analyzed with NMR spectroscopy in solution and revealed a wide range of structural variability for these proteins. Thus, the ID of Kv3.4 was found to exhibit well defined and compact folding although the backbone lacks secondary structural elements. In contrast, the ID from Shaker B and the inactivating N terminus of Kv1.1 (amino acids 1-62) showed no uniquely folded structure but rather behaved like random-coil peptides (16-19).

To gain structural insight into KCNMB2-mediated inactivation of BK channels, we investigated the solution structure and function of the hydrophilic N terminus of KCNMB2 using NMR spectroscopy and giant patch clamp recording on a synthetic peptide (BK₂N) corresponding to the entire cytoplasmic N terminus of KCNMB2 (amino acids 1-45).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electrophysiology-- BK channels were expressed heterologously in Xenopus oocytes as described elsewhere (20). Giant patch recordings were made at room temperature (~23 °C) 3-7 days after injection of capped cRNA encoding hBK (GenBank^TM accession no. U23767). Pipettes used were made from thick-walled borosilicate glass, had resistances of 0.3-0.6 megaohms (tip diameter of about 20 µm), and were filled with (in mM) 5 KOH, 115 NaOH, 10 HEPES, and 0.5 CaCl₂, pH adjusted to 7.2 with MES. Currents were sampled at 10 kHz and corrected for capacitative transients with an EPC9 amplifier (HEKA electronics, Lamprecht, Germany) with the analog filter set to 3 kHz (3 db).

The fast application system used is described elsewhere (17) and allowed for a complete solution exchange in less than 2 ms. BK₂N was dissolved in K_int solution and applied via one barrel of the application system. K_int was composed as follows (in mM): 119 KOH, 1 KCl, 10 HEPES, 1 EGTA, pH adjusted to 7.2 with MES. The amount of CaCl₂ required to yield a free Ca²⁺ concentration of 10 µM was calculated according to Fabiato (36) and added to the EGTA solution under pH meter control. Thereafter, pH was readjusted to 7.2 with KOH.

Rates of inactivation were determined as described previously (17). Briefly, k_off was determined from the time constant of the wash-off (_off) as k_off = 1/_off. k_on was then calculated as k_on = (1/_on  k_off)/[peptide], with _on the time constant for wash-in. Affinity for the peptide-receptor interaction was calculated as k_off/k_on. All values throughout the paper are given as mean ± S.D. of n experiments.

Peptide Synthesis and Sample Preparation-- The BK₂N protein was synthesized by standard solid-phase synthesis and purified by high pressure liquid chromatography. The mass was confirmed by mass spectrometry.

5.4 mg of BK₂N were dissolved in 500 µl of 90% H₂O/10% D₂O (v/v), pH 3.0, resulting in a final peptide concentration of 2 mM. To verify structural properties under physiological conditions, NMR experiments were carried out on BK₂N dissolved in physiological salt solution (90 mM KCl, 10 mM KH₂PO₄, 2 mM MgCl₂) at pH 6.0. All NMR samples contained 2,2-dimethyl-2-silapentane-5-sulfonate as the internal standard for ¹H chemical shift referencing.

NMR Spectroscopy-- Homonuclear NMR spectra were acquired on a Bruker Avance 600 spectrometer at either 293 or 288 K with a spectral window of 11.5 ppm. Standard pulse sequences were used to record NOESY (21) (mixing times between 100 and 250 ms), CLEAN-TOCSY (22) (isotropic mixing time of 80 ms), and DQF-COSY (23) spectra with 4096 data points in F₂ and 512 increments in F₁. All two-dimensional ¹H NMR spectra employed the method of time-proportional phase incrementation for quadrature detection in the F₁ dimension (24). Water suppression was achieved either by presaturation or by the WATERGATE technique (25).

NMR data were processed with the Bruker XWINNMR software using shifted squared sine window functions prior to Fourier transformation. The final matrix size was 4096 × 1024, except for the DQF-COSY spectrum, which was transformed to 16384 × 1024 (corresponding to a digital resolution of 0.42 Hz/point in the F₂ dimension) to extract ³J_HNH coupling constants through a fit of the COSY cross-peaks to two antiphase Lorentzian lines. The programs AURELIA (26) and XEASY (27) were used for analysis of two-dimensional spectra.

Structure Determination-- NOE distance constraints were derived from a 250-ms NOESY spectrum in H₂O and a 200-ms NOESY spectrum in D₂O solution, both recorded at 288 K, pH 3.0. Unambiguously assigned NOESY cross-peaks were integrated manually with XEASY, and the resulting volumes were converted into proton-proton upper distance limits with the program CALIBA (28) using five different classes of NOEs. Constraints for the backbone dihedral angle  were obtained from the ³J_HNH coupling constants  6 Hz. In these cases, a  angle between 85° and 35° was imposed. Structures of BK₂N were calculated with the program DYANA (version 1.5) (29) employing a simulated annealing algorithm in the torsion angle space. Structures from preliminary DYANA calculations were used to recalibrate the distance restraints and to obtain stereospecific assignments by the GLOMSA (28) routine within DYANA. The final family of structures was generated in a calculation with 300 random starting structures and 9000 annealing steps. 30 structures with target function values < 0.92 Å² and no violations of dihedral angle constraints > 5° were obtained. The 24 structures without NOE violations > 0.4 Å were selected for further analysis.

Visualization of structures and preparation of figures were done with the program MOLMOL (30).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BK₂N Inactivated BK Channels in a Ball-like Manner-- The functional characteristics of BK₂N were tested in inside-out patches from Xenopus oocytes expressing non-inactivating homomeric BK channels. As shown in Fig. 1A, BK₂N induced rapid inactivation of the BK -subunit when present at the cytoplasmic side of the patch. Moreover, BK₂N-mediated inactivation occurred only at open channels. Despite the long-lasting presence of BK₂N, channels first opened upon depolarization before they were inactivated by the peptide (Fig. 1, A and B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Functional characteristics of BK2N. A, inactivation of homomeric human BK channels (BK) by 27 µM BK2N continuously present at the cytoplasmic side of a giant inside-out patch. Channels were activated by voltage steps from 100 mV to 10 or 40 mV at a [Ca2+]i of 10 µM. Time and current scaling were as indicated; intra- and extracellular K+ concentrations were 120 and 5 mM, respectively. B, time constant of channel inactivation mediated by 27 µM BK2N as a function of the transmembrane voltage. Data are mean ± S.D. from five experiments. The continuous line represents the fit of the equation A0 + A1·exp[V/] to the data with a value for  of 117.1 mV. Inset, current response to the transmembrane voltage stepped from 100 mV to potentials between 80 and 80 mV with 27 µM BK2N and 10 µM Ca2+ present on the cytoplasmic side of the patch. C, Piezo-driven fast application of 27 µM BK2N to open BK channels as indicated by the horizontal bar. Note the slow dissociation of BK2N from the channel. D, rates of the inactivation process mediated by BK2N. The rates determined for the well structured Kv3.4-ID and the random-coil Kv1.1 N terminus (both at non-inactivating Kv1.1 channels) are given for comparison (19). Values are mean ± S.D. of eight experiments.

The time course of inactivation was strongly dependent on the BK₂N concentration (not shown) and exhibited mild voltage dependence (Fig. 1B). Thus, the time constant as obtained from a monoexponential fitted to the current decay (_inact) changes e-fold with a change in membrane potential of 117 mV, which is equivalent to a valence (z) of 0.21.

These results suggested that very similar to IDs derived from Kv or Kv1.1 subunits, BK₂N blocks BK channels in a "ball-like manner" via interaction with a receptor site on the -subunit that becomes accessible once the channel is in the open state.

Therefore, interaction between BK₂N and the channel -subunit was more closely investigated by the "fast application" technique. This technique allows for complete solution exchanges at inside-out patches in less than 2 ms and enables separate determination for on- and off-rates of channel-peptide interaction (17). Fig. 1C shows rapid application and wash-off of BK₂N at a concentration of 27 µM. Channels were activated prior to peptide application by a voltage-step to 0 mV at a [Ca²⁺]_i of 10 µM. Inactivation occurred with a time constant of 17 ms (16.9 ± 1.5 ms, n = 8), identical to that induced by the continuously present BK₂N (Fig. 1B). Wash-off of BK₂N, which should reflect unbinding of the peptide from the receptor, exhibited a time constant of 850 ms (851.5 ± 69.3 ms, n = 8) and could be well fitted with a monoexponential (Fig. 1C). This was an indication that interaction between BK₂N and its receptor on the -subunit could be described as a bimolecular reaction as suggested (11) with on- and off-rates (k_on, k_off) of 2.0·10⁶ (Ms)¹ and 1.2 s¹ (Fig. 1D), respectively. The affinity (IC₅₀) of BK₂N for its receptor as calculated from these rates is 0.59 µM (Fig. 1D), which is very similar to the value obtained from a steady-state concentration-inhibition relationship (not shown).

Interestingly, a comparison among the inactivation rates of various IDs shows that k_off of BK₂N closely resembles that of the compactly folded Kv3.4-ID, whereas k_on of BK₂N is very similar to that of Kv1.1 or the Shaker B-ID, IDs that both lack ordered three-dimensional structure in solution (Fig. 1D and Ref. 19). Next we investigated the structural properties of BK₂N in solution with NMR spectroscopy.

Assignment and NOE Connectivities of BK₂N-- NMR experiments were performed under various conditions in aqueous solution at pH 3.0 and in a physiological salt solution at pH 6.0 (see "Materials and Methods"). The ¹H NMR resonances of BK₂N were completely assigned by two-dimensional NMR methods in the low pH solution and verified under physiological salt and pH conditions.

As illustrated in Fig. 2A, NOE contacts between nonadjacent amino acids (i,i+x) indicative for structured domains were only observed on the sequence stretch roughly extending from Ser¹⁰ to Leu³¹. This "core domain" exhibited NOE patterns typically observed with -helices. Thus, connectivities between the -proton of one amino acid and the amide (N(i,i+3)) or -proton ((i,i+3)) of the third amino acid following were observed. Most residues throughout this stretch show sequential contacts between backbone amide protons (dNN NOEs). Moreover, ³J_HNH coupling constants were determined in this region for 8 of 22 residues. Five of these J-couplings showed values between 5.3 and 6 Hz, indicative of helical conformations, and three were between 6 and 8 Hz.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   NMR data of BK2N in aqueous solution. A, NOE connectivities and J-coupling constants observed for BK2N in aqueous solution. Sequential and medium range NOEs are shown as a function of the amino acid sequence; the intensity of NOEs is reflected by the line thickness. Filled circles represent 3JHNH coupling constants between 5 and 6 Hz, and open circles represent coupling constants between 6 and 8 Hz. B, upper panel, deviation of H chemical shifts from random-coil values (31). H was calculated as the difference between the experimentally determined H and the random-coil H. Lower panel, bar diagram of NOE constraints by residue. Intraresidual contacts are shown in gray, sequential contacts in white, and medium range contacts in black.

The NOE-based indication of secondary structural elements in BK₂N was corroborated by results of an H/D exchange experiment, where one-dimensional spectra were recorded 15, 30, 45, and 65 min after dissolving lyophilized BK₂N in D₂O. Thus, a number of amide protons (HN) including those of Ile²¹, Gln²³, Ile²⁵, Asp²⁹, and Leu³¹ were identified in the first and second one-dimensional spectrum (see supplemental material). Resonances of the HNs of Ile²⁵ and Leu³¹ were present even in the spectrum recorded 65 min after dissolution, indicating significant protection from exchange with the solvent as would be expected for hydrogen bonding in a helical conformation. This view is further supported by the deviations of the -proton chemical shifts from random-coil values (31). As shown in Fig. 2B (upper panel), the -protons of all residues in the core region are shifted up-field as typically seen in helical structures (32).

Together, the NOE pattern, the H/D exchange, the J-couplings, and the chemical shifts suggest that BK₂N consists of an ordered mostly helical core domain flanked by flexible N and C termini.

Structure of BK₂N in Solution-- A total of 728 experimentally determined NOE constraints (average of 16.2/residue; Fig. 2B, lower panel) together with the restraints for dihedral angles and stereospecific assignment of protons were used to calculate the solution structure of BK₂N (Table I). After structure calculations using the simulated annealing protocol of DYANA (29) in the torsion angle space 24 structures with the lowest values of the target function and without NOE violations of >0.4 Å were selected as the final family of BK₂N structures (for structural statistics see Table I).

View this table: [in this window] [in a new window]   Table I Structural statistics of BK2N

Fig. 3 shows 18 representatives of this family of best structures, superimposed either between residues 10 and 17 (Fig. 3A) or residues 18 and 31 (Fig. 3B). Both superpositions display reasonable convergence with similar r.m.s. deviation values to the mean structure (0.65 ± 0.22 and 0.48 ± 0.27 Å for the backbone atoms in Fig. 3, A and B, respectively), whereas superposition on the entire range was not meaningful because of the divergent orientation of residues Glu¹⁷Arg¹⁹ that connect both stretches. The Glu¹⁷Arg¹⁹ linker thus divides BK₂N into an N- and a C-terminal domain. The N-terminal domain consists of a disordered part made up of residues 1-10 and a loop-helix motif formed by amino acids Ser¹¹Asp¹⁶ (Fig. 3A). Superposition of residues 10-16 revealed r.m.s. deviations from the mean structure of 0.60 ± 0.17 Å for backbone atoms and of 1.14 ± 0.17 Å for all atoms. The C-terminal domain is made up of an extended helical structure formed by residues 20-31 and a flexible C terminus (residues 32-45; Fig. 3B). Within the helical structure, residues 22-30 form a regular -helix that is preceded by one turn of a 3₁₀-helix (Fig. 3B). Superposition of structures over the range of the helix domain results in r.m.s. deviations of 0.43 ± 0.26 Å for backbone atoms and of 0.90 ± 0.25 Å for all atoms.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Solution structure of BK2N exhibits two highly ordered domains. A, backbone superposition (N, C, and C atoms) of the family of best structures (with lowest target function) of BK2N between residues 10 and 17 (highlighted in red). B, backbone superposition shown as in A but between residues 18 and 31 (highlighted in red). Both panels depict 18 of the 24 best structures of BK2N (see text). The N and C termini are indicated.

When correlated with functional properties, it is only the N-terminal domain that is required for occlusion of the channel pore, as seen in experiments with this domain fused to the KCNMB1 subunit or applied to BK channels as a synthetic peptide (11). Accordingly, this domain was termed the ball domain (Fig. 4). In contrast, the C-terminal domain, which links the ball to the transmembrane segment of KCNMB2, may be regarded as the chain domain (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Ball and chain domains of BK2N. N-terminal 34 residues of the best BK2N structure in a ribbon representation emphasizing the secondary structural elements between residues 11-17 and 20-30. Functional analysis delineated residues 1-18 as the pore-occluding ball domain, and residues 20-45 represent the chain domain linking the ball to the transmembrane core of the KCNMB2 protein. To emphasize the flexibility of the very N terminus (residues 1-10), two representatives of the family in Fig. 3 are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BK₂N inactivates BK channels with characteristics known from the "pore plug-in" described for inactivation domains of Kv-type K⁺ channels. Accordingly, BK₂N-mediated pore occlusion exhibits shallow voltage dependence and is competed by the pore-blocking agent TEA (11, 13). As determined from NMR experiments, BK₂N presents with a unique solution structure; it consists of two domains connected by a flexible linker, the ball domain, made up of a disordered N terminus anchored at a loop-helix motif, and the chain domain, a 4-turn helix with an unfolded region at its C terminus.

As shown in Fig. 1B, the structure of BK₂N is accompanied by functional properties that are unique with respect to those of Kv-derived IDs. Thus, the association rate of BK₂N with its receptor on the channel is very similar to that observed with the unstructured IDs of Kv1.1 or Shaker B, but considerably slower than that determined for the compactly folded Kv3.4-ID (17, 19, 33). The dissociation rate of BK₂N, on the other hand, is more than 10-fold lower than that of Kv1.1-ID or the ID of Shaker B and even about 2-fold lower than that of Kv3.4-ID.

These correlations between structural and functional properties are consistent with our earlier observations that well ordered IDs exhibit a faster k_on and a much slower k_off than unfolded domains (17, 19). The latter seems to be caused either by the higher number of molecular contacts (hydrogen bonds, etc.) formed between the folded domain and the receptor or by the higher flexibility of the unfolded IDs, which destabilizes the ID-receptor interaction.

Together with the observation that the actual pore block is realized by the N-terminal 19 or 26 residues (11), BK₂N-mediated inactivation may be imagined to occur as follows. The ball domain (Fig. 4) will approach the open channel and, in a second step, bind to its receptor, which finally results in occlusion of the channel pore. Channel approach and binding are reflected by k_on and are determined by the flexible part of the ball domain as suggested recently for Kv1.1-mediated inactivation (34). Unbinding of the ID from its receptor, as reflected by k_off, should be controlled by the structured part of the ball domain. As k_off of BK₂N is the lowest of all ID peptides investigated to date, the ball-receptor interaction must be particularly strong. Interestingly, the structured region of the ball domain contains a cluster of charged residues suggesting that hydrophilic interactions may be an important determinant for the BK₂N-BK interaction. This premise would be in line with work by Toro et al. (35) who investigated the interaction between the ID from Shaker B with BK channels and concluded that the ID receptor of BK channels in the inner vestibule of the channel may contain hydrophilic residues and a "pocket" that favors binding of helical structures.

The molecular identity of the BK ID receptor, however, must remain open at this point as well as the question of how far BK₂N enters the channel pore and whether interactions between charges on the ball domain and the channel wall are involved in ID receptor interaction.

	ACKNOWLEDGEMENT

We are indebted to Otogene AG (Tübingen) for access to the NMR spectrometer.

	FOOTNOTES

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Fa 332/3-1), the Federal Ministry of Education, Science, Research and Technology (Fö.01KS9602), and the Interdisciplinary Center of Clinical Research, Tübingen (Project IA4) (to B. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material on the 1D spectra of the H/D exchange experiments.

The atomic coordinates and chemical shifts of the final 24 structures (code 1JO6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) and the BioMagResBank (accession number 5092).

^§ Current address: Dept. of Physiology II, University of Freiburg, Hermann-Herder-Str. 7, Freiburg, Germany.

^¶ To whom correspondence should be addressed: Ob dem Himmelreich 7, D-72074 Tübingen, Germany. Tel.: 49-7071-2977363; Fax: 49-7071-87815; E-mail: detlef.bentrop@uni-tuebingen.de.

Published, JBC Papers in Press, August 21, 2001, DOI 10.1074/jbc.M107118200

	ABBREVIATIONS

The abbreviations used are: BK, large conductance Ca²⁺ and voltage-dependent K⁺ channel; Kv, superfamily of voltage-dependent K⁺ channels; BK₂N, synthetic peptide covering the N-terminal 45 amino acids of the ₂-subunit of the human BK channel (human KCNMB2); NOE, nuclear Overhauser effect; ID, inactivation domain; MES, 4-morpholineethanesulfonic acid; r.m.s., root mean square.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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