N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit*

Background: The large-conductance Ca2+-activated K+ channel (BKCa) is an important regulator of membrane excitability. Results: N-terminal isoforms of the BKCa channel are differentially modulated by the regulatory β1-subunit. Conclusion: Expression of different N-terminal isoforms is a novel mechanism of BKCa channel regulation. Significance: Elucidating the modulation of BKCa activity by β1 provides a new understanding of ion channel physiology. The large-conductance Ca2+-activated K+ (BKCa) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BKCa α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BKCa α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca2+. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BKCa channel activity in cells, highlighting a novel mechanism of BKCa channel regulation.

The large-conductance Ca 2؉ -activated K ؉ (BK Ca ) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK Ca ␣-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the ␣-subunit are required for modulation by auxiliary ␤1-subunits, we examined whether ␤1 differentially modulates the N-terminal BK Ca ␣-subunit isoforms. In the absence of ␤1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas ␤1 did not modulate the voltage-activation curve of MSSN, ␤1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 M and 100 M Ca 2؉ . The ␤1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the ␤1-subunit may be due to the differential effect of ␤1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related ␤2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to ␤1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK Ca channel activity in cells, highlighting a novel mechanism of BK Ca channel regulation.
The large-conductance voltage-and Ca 2ϩ -activated K ϩ channel (BK Ca ) 2 is a key regulator of membrane excitability in a wide variety of cells such as neurons, smooth muscle, chromaffin cells, and immune cells (1)(2)(3)(4)(5). BK Ca channel activation integrates both membrane depolarization and increases in intracellular Ca 2ϩ to control membrane excitability (6). Although a single gene (KCNMA1) encodes the BK Ca channel ␣-subunit, alternative splicing produces an array of BK Ca isoforms that respond to a variety of modulators in tissue-specific manners (4,(7)(8)(9). Despite the high degree of splicing, all BK Ca ␣-subunits are composed of seven conserved transmembrane domains (S0 through S6) and an extracellular N terminus. The N terminus and first transmembrane domain (S0) of the ␣-subunit are required for association with the auxiliary ␤1-subunit (10,11), which is an important source of variable regulation for BK Ca channel function (12)(13)(14).
Three possible translation initiation sites have been identified in the BK Ca ␣-subunit N-terminal sequence (15,16). Heterologous expression studies have used the third initiation site (methionine at position 66), starting with amino acid sequence MDAL, as the canonical human BK Ca channel ␣-subunit to investigate ␤-subunit modulation (10,14,17). Other studies of the mouse (13,18) or rat (9) BK Ca channel have also used N-terminal truncated isoforms that start at the second or third translation initiation sites (starting with MSSN or MDAL, respectively). The full-length transcript, which can encode an isoform that starts with MANG, has been isolated from human smooth muscle (15), but whether the ␤1-subunit modulates this isoform of the human BK Ca ␣-subunit has not been extensively investigated. Because the N-terminal region and first transmembrane domain (S0) are necessary for ␤1-subunit modulation (10,11), we hypothesized that these three N-terminal isoforms of the BK Ca ␣-subunit may be differentially modulated by the accessory ␤1-subunit. Here, we report that the ␤1-subunit differentially modulates the activation of the MANG, MSSN, and MDAL isoforms. We show that ␤1 modulates the voltage activation of the N-terminally extended isoforms less than that of the MDAL isoform. This effect may reflect differential modulation of channel kinetics of the three isoforms by ␤1. By contrast, the ␤2-subunit modulated voltage activation of MDAL, MSSN, and MANG isoforms to similar extents. These results suggest a new mechanism of fine-tuning the BK Ca channel activity that depends on the expression of the N-terminal isoforms and their modulation by the ␤1-subunit.

EXPERIMENTAL PROCEDURES
Tissue Collection and Cloning of BK Ca Isoforms-Non-pregnant human uteri were obtained from the Cooperative Human Tissue Network (Midwestern Division, Columbus, OH). Total RNA from human myometrium was reverse transcribed by using the First-strand RT-PCR kit (Stratagene, La Jolla, CA). The MANG (GenBank TM accession no. BC137137.1) and MSSN isoforms of the BK Ca ␣-subunit were isolated from human myometrium by using sense primers 5Ј-ATGGCAAA-TGGTGGCGGC-3Ј and 5Ј-ATGAGTAGCAATATCCAC-3Ј, respectively, with the antisense primer 5Ј-CCCAGTAGAGT-CGTACTT-3Ј. The MDAL isoform (GenBank TM accession no. U11058.2) was subcloned by PCR using the MANG isoform as a template.
cDNA Constructs-All BK Ca ␣-subunit N-terminal isoforms were inserted into the pCMV site of a pBudCE4.1 plasmid vector (Invitrogen) containing an optimal Kozak sequence (GAC-CACC) upstream of the start codon and including the mCherry reporter in the EF1-␣ site. The cDNA encoding the human ␤1-subunit (GenBank TM accession no. U25138.1) was cloned into the EF1-␣ site of pBudCE4.1; eGFP was cloned by PCR into the pCMV site as a reporter. The ␤2-subunit construct with its N terminus deleted (⌬2-20, ␤2ND) (19) was kindly provided by Dr. Jianmin Cui (Washington University in St. Louis) and was cloned into the EF1-␣ site of pBudCE4.1; eGFP was used as a reporter. Plasmid DNAs for transfection were isolated with a Plasmid Maxi kit (Qiagen, Hilden, Germany).
Cell Culture and Transfection-Human embryonic kidney (HEK) 293T cells were grown to 60 -80% confluency in DMEM/F12 supplemented with 10% fetal bovine serum and 50 g/ml gentamicin (all from Invitrogen). Cells were transiently transfected with constructs expressing the human BK Ca channel N-terminal isoforms. Another set of cells was co-transfected with the BK Ca ␣-subunit isoforms and the ␤1-subunit (in a 1:1 or 1:10 molar ratio) or the ␤2ND-subunit. All transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were used in subsequent experiments 24 -48 h post-transfection.
Electrophysiology-Single-channel recordings in the insideout configuration were performed at room temperature in a bath solution containing the following: 140 mM KCl, 20 mM KOH, 10 mM HEPES, 5 mM (H)EDTA, and 10 M or 100 M free-Ca 2ϩ (pH 7.2 with HCl). Free Ca 2ϩ concentration was measured by using a Ca 2ϩ -sensitive electrode (Thermo Fisher Scientific, Waltham, MA). Pipette solution contained the following: 140 mM KCl, 20 mM KOH, 2 mM MgCl 2 , and 10 mM HEPES (pH 7.4). Single-channel currents were recorded at a sampling rate of 100 kHz and filtered at 5 kHz by using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA).
Currents were evoked with 10 mV voltage steps (1000-ms duration) from Ϫ160 to ϩ120 mV, from a holding potential of 0 mV by using pCLAMP software (version 10, Molecular Devices). This protocol was repeated at least three times on each patch with 10 M or 100 M Ca 2ϩ in the bath. For analysis purposes, recordings on the same patch were concatenated, attaining at least a 3-s length recording from each voltage pulse. Mean open probability (P o ) and unitary current (i) values were calculated by using pCLAMP software. Patches containing three or fewer channels were used for P o analysis; only patches containing one channel were used for i measurements. P o was plotted against membrane potential (V) and fitted to a Boltzmann function, to determine half-maximal activation voltage (V 0.5 ) and effective charge (z) values for each experiment by using Graph Pad software (San Diego, CA). Unitary conductance (␥) values were obtained from plotting the i-V relation (from Ϫ60 to ϩ60 mV) and fitting to a linear regression; the slope of that regression was calculated as ␥ for each experiment. For analysis of open and closed dwell times of the channel (MANG, MSSN, or MDAL) in the presence or absence of ␤1-subunit, inside-out patches containing only one channel were held at Ϫ20 mV for 3 min with 10 M Ca 2ϩ in the bath. Open and closed dwell times histograms were plotted in log-bin time scales and fitted with double exponential functions to obtain time constants () and relative distribution (P) of the data under the curve by using pCLAMP software. Burst analyses were performed on the same recordings as the dwell time analyses; bursts correspond to openings separated by a minimal closed interval defined as critical closed ( crit ). crit was estimated as the second or third component of a five component exponential function fitting within the closed dwell time histogram, as described previously (20). To ensure a clear estimation of crit , only recordings with a P o value less than 0.8 were included in the burst analysis.
Statistical Analysis-Data obtained were subjected to nonparametric Mann-Whitney U test by using Graph Pad software. A p value Ͻ 0.05 was considered significant. All data are presented as mean Ϯ S.E.

BK Ca N-terminal Isoforms Have Distinct Modes of Regulation
by the Auxiliary ␤1-subunit-Previous studies identified three possible translation initiation sites in the BK Ca ␣-subunit N-terminal sequence (15,16). We cloned the longer N-terminal BK Ca isoform, with amino acid sequence starting at MANG and the isoform starting at MSSN from human myometrium samples. Then, we used PCR to subclone the shorter N-terminal form starting at MDAL (Fig. 1); all constructs contained an optimal Kozak sequence at the 5Ј end to optimize expression.
To test the functional properties of the N-terminal isoforms of the BK Ca ␣-subunit, we expressed each construct individually in HEK293T cells and evaluated their voltage-and Ca 2ϩ -dependent currents. Inside-out patch-clamp recordings showed a similar voltage-and Ca 2ϩ -dependence of MANG, MSSN, and MDAL isoforms; all three channels showed a volt-age-induced increase in P o (Fig. 2). Changes in Ca 2ϩ concentration in the bath from 10 M to 100 M induced similar leftward shifts in the voltage-activation curves of MANG, MSSN, and MDAL (ϳ50 mV, ϳ44 mV, and ϳ51 mV, respectively, Fig. 2 and Table 1). In addition to P o , unitary conductances were comparable between these isoforms (Fig. 2I). Co-expression of the ␤1-subunit elicited a significant leftward shift in the voltageactivation curve of MDAL (ϳ47 and ϳ53 mV at 10 and 100 M Ca 2ϩ , respectively, Fig. 2 and Table 1). Although the ␤1-subunit also shifted voltage-activation curves of the MANG isoform to the left, the shifts were significantly smaller than those of MDAL (ϳ24 and ϳ25 mV at 10 and 100 M Ca 2ϩ , respectively, Fig. 2 and Table 1). In contrast, co-expression of ␤1 did not significantly shift the voltage-activation of the MSSN isoform ( Fig. 2 and Table 1). The ␤1-subunit reduced unitary conductance only in the MDAL isoform (Fig. 2I). Moreover, the ␤1-subunit did not change the effective charge (z, given by the slope of voltage-activation curves) of any isoform at 10 M or 100 M Ca 2ϩ (Table 1).
We further analyzed the effect of the ␤1-subunit on the MANG, MSSN, and MDAL channel kinetics by recording single channel currents at Ϫ20 mV for 3 min with 10 M Ca 2ϩ in the bath. In the presence of the ␤1-subunit, the open time constants () were increased in MANG and MDAL, whereas only the larger ( 2 ) was increased in MSSN ( Fig. 3 and Table 2). Additionally, ␤1 decreased the of the closed state of both MANG and MDAL isoforms, but increased the of the closed state on MSSN ( Fig. 3 and Table 2). Co-expression of ␤1 significantly increased the burst duration of both MDAL (from 18.0 Ϯ 6.0 ms to 59.3 Ϯ 18.5 ms, p Ͻ 0.05, Fig. 3D) and MSSN (from 23.9 Ϯ 3.6 ms to 52.6 Ϯ 10.2, p Ͻ 0.05, Fig. 3D), but not of MANG (from 11.5 Ϯ 2.8 ms to 17.1 Ϯ 6.9 ms, Fig. 3D). Interestingly, the interburst interval was increased on MANG and MSSN isoforms in the presence of the ␤1 subunit, but not in MDAL (Fig. 3E), indicating that the ␤1-induced stabilization of the burst state was different between these isoforms (Fig. 3F). Together, these observations suggest that the extended N-terminal sequence in the MANG isoform reduces, and in the case of MSSN abolishes, the ␤1-induced changes in voltage dependence of channel activation, which might be due, in part, to a differential ␤1-dependent modulation of channel kinetics.
A typical BK Ca channel is thought to have a stoichiometry of 4␣:4␤1-subunits (21). However, the relative expression of each of these proteins has been reported to influence the overall effect of the ␤1-subunit on BK Ca currents; binding of each ␤1 molecule can sequentially shift the V 0.5 of the channel (22). Therefore, it is feasible that the intermediate effect of ␤1 observed in the MANG isoform was due to fewer ␤1-subunit molecules binding to the ␣-subunit when expressed in 1:1 ratio. To address this possibility, we used an ␣:␤1 cDNA ratio of 1:10 to express the MANG and MDAL isoforms in the presence of saturating concentrations of ␤1. The resulting voltage activation curves for both MANG and MDAL were similar to those observed when a 1:1 cDNA ratio was used (Fig. 4). Thus, the reduced ␤1-induced leftward shift of MANG voltage-activation curves was not due to fewer ␤1-subunits binding to this isoform.
All Three N-terminal Isoforms Are Similarly Modulated by the ␤2-subunit-Similar to ␤1, the related ␤2-subunit increases BK Ca channel Ca 2ϩ sensitivity (23), although it is likely that modulation by these subunits occurs by different mechanisms (19,24,25). To assess whether the significantly attenuated ␤1-induced leftward shift of the voltage-activation curve observed in the MANG isoform, and the lack of effect on the MSSN isoform, were specific for the ␤1-subunit, we tested the modulation of MANG, MSSN, and MDAL by ␤2. Because ␤2 inactivates the BK Ca ␣-subunit channel currents by N-type inactivation, we used a truncated form of the ␤2-subunit (␤2ND) that does not inactivate the channel (19,23). We observed a significant leftward shift in the voltage-activation curves at two Ca 2ϩ concentrations in cells co-expressing any of the ␣-subunit isoforms with ␤2ND as compared with those expressing ␣-subunit alone (curves were shifted to the left by ϳ50 mV and ϳ66 mV for MANG, by ϳ23 mV and ϳ58 mV for MSSN, and by ϳ59 mV and ϳ57 mV for MDAL, in the presence of 10 and 100 M Ca 2ϩ , respectively, Fig. 5). Hence, the decreased shift in channel activation by the extended N terminus of the MANG and MSSN isoforms is specific for the ␤1-subunit.

DISCUSSION
The BK Ca channel plays an important role in regulating the membrane potential of excitable cells. Association of the poreforming ␣-subunit of the BK Ca channel with distinct auxiliary subunits (␤1-␤4 or the recently described ␥1-␥4) is a significant form of channel modulation (17,26). Accordingly, interaction of the ␣-subunit with the ␤1-subunit, which predomi- nates in smooth muscle, confers increased Ca 2ϩ sensitivity and decreased voltage-dependence to the BK Ca channel (12,13). Both the N-terminal region and the S0 transmembrane domain of the BK Ca ␣-subunit are essential for both its interaction with the auxiliary ␤1-subunit (10, 11) and for ␤2-subunit modulation (19). In this work, we studied the properties of three differ-ent N-terminal isoforms (MANG, MSSN, and MDAL) and how they are modulated by the auxiliary ␤1and ␤2-subunits. We observed that whereas the voltage-activation curve of MDAL was shifted to the left by ϳ50 mV, that of MANG was only shifted by ϳ25 mV, and that of the MSSN isoform was not affected by the ␤1-subunit.  The diminished ␤1-induced shift in voltage-activation curves observed in the MANG isoform, and the lack of modulation in MSSN, were reflected in the modulation of channel kinetics. We observed that the ␤1-subunit modulated channel kinetics of all three BK Ca ␣-subunit N-terminal isoforms, as evidenced by increases in open dwell times. We also observed that the closed dwell times of MANG and MDAL decreased, whereas the closed dwell times of MSSN increased; these data are consistent with other reports showing that the ␤1-subunit modifies the kinetics of the channel by stabilizing its open state (14,18). The ␤1-subunit also increases the apparent Ca 2ϩ sensitivity of the channel by increasing burst duration (20). Analysis of single-channel kinetics revealed that both MDAL and MSSN isoforms were maintained in the bursting state by ␤1, as indicated by an increased burst duration, but the burst duration of the MANG isoform was not affected by the ␤1-subunit. Furthermore, ␤1 increased the interval between bursts of MANG and MSSN isoforms but did not change the interval of the

TABLE 2 Effect of ␤1-subunit on the open and closed dwell-times of different BK Ca ␣-subunit N-terminal isoforms
Time constants ( 1 and 2 ) are expressed in milliseconds. P 1 and P 2 are relative distributions of data under curves used to fit the results shown in Fig. 3. MDAL isoform. These observations (summarized in Table 3   The association between BK Ca ␣and its ␤1-subunit plays an important role in multiple tissues, most notably in regulating smooth muscle contractility (12,(27)(28)(29)(30)(31). For example, the ␤1-subunit controls arterial tone in resistance arteries (29) and contractility in the urinary bladder (31). Because the interaction between BK Ca ␣ and ␤1 seems to be important for human myometrial contractility (32,33), and both subunits are abundantly expressed in this tissue (34), we explored the presence of the different N-terminal isoforms in smooth muscle tissue derived from human myometrium obtained from non-laboring pregnant women at term. However, detection of peptides corresponding to the various N termini by using mass spectrometry was unsuccessful (data not shown), likely because the low complexity of the sequence between amino acids Met-1 and Met-66 (polyglycine and polyserine stretches, Fig. 1) complicates the detection of these peptides by mass spectrometry. Further studies, such as N-terminal protein sequencing or development of specific antibodies targeted to the N-terminal region of the BK Ca ␣-subunit, will be necessary to determine the endogenous expression of these isoforms.

Isoform
Our data suggest that the additional N-terminal sequence in the MANG isoform might dampen the effects of the ␤1-subunit on channel activation by a specific allosteric mechanism rather than by hampering protein-protein interaction. The case of MSSN seems to be more complex; extending the N-terminal sequence by 41 amino acids completely abolished the modulation by ␤1 of the voltage-dependent activation observed with MDAL, but adding 24 more amino acids partially restored this modulation, as observed with the MANG isoform. The secondary structure of this N-terminal sequence is not clear because of its low complexity poly-glycine and poly-serine stretches (Fig.  1). Further studies aimed at dissecting the length of residues necessary to prevent and restore ␤1 modulation might elucidate the structural determinants of this effect.
Several studies have reported that the ␤1-subunit increases the Ca 2ϩ and voltage sensitivity of recombinant BK Ca channels (9 -11, 13-15, 17, 18, 35-37). However, most reports used an ␣-subunit isoform starting at the third initiation site, the MDAL isoform (10,14,15,17). Although three predicted initiation codons are encoded in the DNA sequence of the ␣-subunit (15,16), neither the first nor second initiation sites (corresponding to MANG and MSSN isoforms, respectively) generate functional channels when expressed in Xenopus laevis oocytes (15). One study demonstrated that expression of functional BK Ca channels might depend on the taxonomic class of the cell line used for heterologous expression. Erxleben et al. (9) described a BK Ca channel in a rat pituitary cell line, starting at MSSN, whose kinetics are slower in the presence of ␤1, but lack the typical change in Ca 2ϩ sensitivity at 1-10 M Ca 2ϩ . Interestingly, a truncated form starting at MDAL restores modulation by the ␤1-subunit. These authors propose that the polyserine stretch between Met-25 and Met-66 is important in blocking Ca 2ϩ sensitivity modulation by ␤1. We found comparable results in that the human MSSN isoform lacked, and the MDAL isoform retained, voltage-dependent activation modulation by the ␤1-subunit. In our study, using a mammalian heterologous expression system, both MANG and MSSN N-terminal isoforms formed functional channels with intrinsic properties indistinguishable from those observed in the MDAL isoform. In addition, expression of the N-terminal isoforms in a mouse fibroblast cell line also generated functional channels, comparable to those expressed in HEK293T cells (data not shown). Thus, expression systems may account for the differences in BK Ca channel N-terminal ␣-subunit function observed between our experimental conditions and those in other reports; however, the underlying mechanism for tissue specific expression of these N-terminal isoforms is still unclear.
The functional complexity of the N-terminal BK Ca isoforms, and how their expression is regulated, has not been fully explored. Alternative translation initiation has been described to modulate the expression and function of N-terminal truncated forms of certain potassium channels: the voltage-gated potassium channel Kv3.3 (38) and the two P-domain potassium channels K 2P 2.1 and K 2P 10.1 (39,40). Alternative translation initiation is a mechanism to regulate protein diversity whereby proteins with different N termini are produced from a single mRNA (41). Alternative translation initiation occurs during translation when the ribosome binds to an initiation codon (AUG) that is not the first cap-proximal in the mRNA coding region, thereby generating N-terminally truncated protein isoforms. Generally, AUG sequences are flanked by Kozak consensus sequences, which facilitate binding of the ribosome to the AUG sequence and thus translation initiation (42). The extent of this facilitation is determined by the relative strength of the ribosome binding given by the Kozak sequence; thus, some sequences will promote binding of the ribosome to a certain initiation site over others (43). In examining the mRNA sequence of the BK Ca channel ␣-subunit, we found that the third initiation codon (Met-66) is flanked by a Kozak sequence stronger than those found in the first or second initiation codon (Met-1 and Met-25, respectively); this might lead to leaky scanning by ribosomes to initiate translation at MDAL. In our study, our N-terminal constructs contained an optimal Kozak consensus sequence (GACCACC) before their respective initiation codons to ensure their optimal heterologous expression. Nonetheless, it is possible that the expression of the MDAL isoform was facilitated by alternative translation initiation, at the expense of expression of MANG or MSSN isoforms; therefore, additional studies are necessary to clarify the molecular mechanisms of this regulation. Our data suggest that the relative expression of N-terminal isoforms can act as a novel mechanism of regulation of BK Ca channel activity by the auxiliary ␤1-subunit. Thus, our results showing the differential activity of BK Ca channel N-terminal isoforms might be relevant to several tissues and pathological processes in which BK Ca channels, and their ␤1-subunits, are involved and could lead to development of specific therapeutic strategies to regulate channel activity.