A New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function*

Large conductance Ca2+-activated K+ (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca2+ entry, and forms a positive feed-back mechanism for Ca2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127–Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158–Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis.

chondrocytes. Under pathophysiological conditions, such as osteoarthritis (OA), histamine release from resident mast cells enhances production of proinflammatory mediators and matrix degrading enzymes (matrix metalloproteinases). Histamine can also alter the proliferation of chondrocytes in articular joints. Specific spatial and temporal patterns of changes in [Ca 2ϩ ] i are considered essential components of disease initiation and progression.
In a well studied model cell of the human chondrocyte, OUMS-27 cells, BK channels (as well as other Ca 2ϩ -activated K ϩ (K Ca ) channels) are functionally expressed. BK channels are involved in enhancement of histamine-induced Ca 2ϩ influx after membrane hyperpolarization (8,10). BK channels also act as mechano-sensing elements in chondrocytes, as judged by their sensitivity to hypo-osmotic challenges (11). The BK channel expression is up-regulated in patients with progressive OA (12). For these reasons, BK channels are thought to be involved in physiological and/or pathological regulation of chondrocyte function.
We have identified a novel BK␣ splice variant (BK␣⌬e2: AB524033.1) in OUMS-27 cells. The present study was undertaken to identify the molecular details of this novel variant and define its functional roles in chondrocytes. Our data demonstrate that this BK␣⌬e2 splice variant can negatively regulate functional expression of BK channels and modulate essential cellular functions, such as cyclooxygenase-2 (COX2) gene expression, in chondrocytes.

Results
Identification of a Novel Splice Variant of BK␣-A novel splice variant of BK␣, which is 54 amino acids shorter than wild-type BK␣ (BK␣WT), was cloned (supplemental Fig. S1) from an OUMS-27 human chondrocyte cell line. This variant lacks a region encoded by exon2 (Glu 127 -Leu 180 ) corresponding to the latter part of S0-S1 linker (Trp 108 -Arg 178 ) and first two amino acid residues (Val 179 and Leu 180 ) of S1 segment (Val 179 -Ser 199 ) (BK␣⌬e2, Fig. 1A). Recently NMR analysis revealed that the S0-S1 loop contains two short ␣-helix (Helix1 and Helix2) (13). As shown in Fig. 1B, the deletion of exon2 does not disrupt the overall hydrophobicity pattern of S1 segment of BK␣⌬e2. Instead, it alters hydrophilic region and the Helix2 in S0-S1 loop.
RT-PCR analysis establishes that this BK␣⌬e2-splice variant is expressed in OUMS-27 cells, human cartilage from both healthy and OA specimens (Fig. 1C, see supplemental Table S1 for primer information). Expression of BK␣⌬e2 mRNA has also been detected in other human tissues, such as heart and lymph node (Fig. 1C). In addition, quantitative real-time PCR (Q-PCR) analysis revealed relatively high expression of BK␣⌬e2 in OUMS-27, a well studied human chondrocyte cell line (relative expression to WT: 0.14 Ϯ 0.03, n ϭ 4) and trachea and aorta from mice (0.08 Ϯ 0.03 and 0.12 Ϯ 0.04, n ϭ 4 each; Fig. 1D).
The BK␣⌬e2 Splice Variant Does Not Exhibit Ion Channel Function-To evaluate whether BK␣⌬e2 can form functional channels, this transcript was labeled with a FLAG tag at its extracellular N terminus and also by mCherry (mCh) at the intracellular C terminus and then transiently expressed in HEK293 cells. BK-mediated currents were assessed using whole-cell patch clamp technique. When the pCa of pipette filling solution was 6.5, cells expressing BK␣WT exhibited paxilline (Pax, a specific BK channel blocker)-sensitive currents; in contrast, BK␣⌬e2-transfected cells did not (Fig. 2, A  and B). As shown in Fig. 2C, the unitary current amplitude (measured using inside-out patch clamp recordings) was 11.5 Ϯ 0.9 pA at ϩ50 mV (4 single channel recordings) in HEK293 cells expressing BK␣WT, but no single channel current was detected from cells expressing BK␣⌬e2 (5 recordings).
In summary, BK␣⌬e2 can form hetero-tetramers with BK␣WT that yield functional channels which have smaller single channel conductance and shorter mean open time than the corresponding homo-tetrameric BK␣WT channels. Based on the conductance shown above, it is apparent that WTϩ⌬e2 #1 corresponds to WT homo-tetramer. However, the stoichiometry in #2 and #3 is unclear.
BK␣ Tetramer Containing One, Two, or Three BK␣⌬e2 Subunits in PM Can Permeate K ϩ -The stoichiometry of functional BK␣WT/BK␣⌬e2 hetero-tetramers was examined based on data from studies using tetraethylammonium (TEA, a BK channel blocker) as a pharmacological tool. It has been reported that when tyrosine residue at 359 in BK␣WT is replaced with valine (BK␣Y359V), the resulting variant is insensitive to TEA (Fig. 3A) (14). When BK␣Y359V-mCh and BK␣WT-GFP are co-expressed in HEK293 cells (WT(Y359V)ϩWT), unitary current amplitude in the presence of 2 mM TEA increased in approximate proportion to the number of BK␣Y359V subunits within a BK␣ tetramer (predicted number of WT(Y359V) and corresponding unitary currents in WT(Y359V)ϩWT: one and 1.4 Ϯ 0.1 pA (4 recordings), two and 3.2 Ϯ 0.1 pA (10 record-ings), three and 6.6 Ϯ 0.2 pA (5 recordings), four and 9.0 Ϯ 0.2 pA (6 recordings) (Fig. 3, B and C, and supplemental Table S3) (14).
In cells co-expressing BK␣Y359V-mCh and BK␣⌬e2-GFP (WT(Y359V)ϩ⌬e2), unitary current distribution was divided into three groups (group 1: 8.9 Ϯ 0.1 pA (12 recordings); group 2: 6.7 Ϯ 0.2 pA (4 recordings); group 3: 2.9 Ϯ 0.2 pA (4 recordings); Fig. 3, B and C, and supplemental Table S3). Then, it was examined to determine if the mean unitary currents of these three groups matched those of the corresponding groups in WT(Y359V)ϩWT cells, respectively (Fig. 3C). Statistical analyses denote distributions of single channel currents are equal between two groups having closest mean values (p Ͼ 0.05 by F-test). There is no significant difference (p Ͼ 0.05 by t test) between each set of three pairs.
Molecular Imaging Shows Hetero-tetramer Formation of BK␣⌬e2 and BK␣WT in PM-To complement and extend our electrophysiological findings, these same BK␣ hetero-tetramer complexes at the surface membrane were analyzed using confocal microscopy. A non-permeabilized (NP) labeling method was used to identify the BK channels that expressed well only in the surface membrane (PM) (3,5). Because the N terminus of BK␣ is located in extracellular space (Fig. 1A), the N termini of both BK␣WT and BK␣⌬e2 were fused with FLAG tag. In addi-FIGURE 2. Co-expression of BK␣⌬e2 with BK␣WT attenuates whole-cell currents and the single-channel conductance. A, whole-cell currents recorded from HEK293 cells expressing BK␣WT or BK␣⌬e2 transcripts. To define the BK current component, a selective blocker 1 M Pax was applied to these cells. B, electrophysiological data showing the relationship between Pax-sensitive current amplitude and activation voltage. The number of experiments is shown in parentheses. C, inside-out patch clamp recordings were used to measure single channel currents due to BK␣WT or BK␣⌬e2 transcripts. D, whole-cell currents in HEK293 cells co-expressing WT-CFP and WT-mCh (WTϩWT) or WT-CFP and ⌬e2-mCh (WTϩ⌬e2). Cells were transfected with cDNA at a ratio (WT:⌬e2) of 1:3. E, current-voltage relationships for the Pax-sensitive currents. F, single channel currents (left) and corresponding amplitude histograms of the currents (right) were obtained from cells expressing both WT and ⌬e2 at ϩ50 mV. In control cells (WTϩWT), these unitary currents show a single distribution pattern that peaks at 11.2 pA. In contrast, the amplitude histogram in WTϩ⌬e2 forms three distinct groups (#1, 11.1 pA; #2, 9.7 pA; #3, 3.5 pA). Cells were transfected with cDNA at a ratio (WT:⌬e2) of 1:1. G, single channel conductance in WTϩWT (8 recordings from 8 patches) and WTϩ⌬e2 (#1, #2, and #3, 30 recordings from 27 patches) are summarized. **, p Ͻ 0.01 versus WTϩWT; ##, p Ͻ 0.01 versus #1; $$, p Ͻ 0.01 versus #2. See also supplemental Table S2. tion, the C termini were tagged with mCh. In HEK293 cells expressing BK␣WT, the anti-FLAG antibody detected extracellular FLAG tag fused to BK␣WT (Alexa488, Fig. 4A). The anti-FLAG antibody did not recognize FLAG tag conjugated to BK␣⌬e2; however, after membrane permeabilization, it did so. The ratio of anti-FLAG positive to mCh-positive cells, which Each potential hetero-tetramer complex in a cell expressing WT(Y359V)ϩWT is also illustrated in the current traces. Note that unitary current distribution was divided into three distinct groups (Groups 1-3) in cells expressing WT(Y359V)ϩ⌬e2. C, scatter plots of the unitary current amplitude recorded in cells expressing WT(Y359V)ϩWT (25 recordings from 12 patches) or cells expressing WT(Y359V)ϩ⌬e2 (20 recordings from 16 patches). There was no significant difference in the distribution (p Ͼ 0.05 by F test) and mean value (n.s., p Ͼ 0.05 by t test) between the two types of cells. Predicted model of hetero-or homo-tetramer complexes are shown in both sides of the scatter plots. D, frequency distribution (%) of channels including the predicted number of WT(Y359V) within the single tetramer complex. Here the number of recordings that contained single channel currents due to each complex of BK␣ tetramer were counted. The total number of BK␣ tetramers within each patch could not be confirmed in this study. See "Experimental Procedures" for details. E, only when membrane patches were excised from cells exhibiting very strong fluorescence derived from ⌬e2-GFP, unitary currents smaller than 2 pA were detected. Cells were transfected with cDNA at a ratio (WT(Y359V)-mCh:⌬e2-GFP) of 1:1. F, the unitary currents were compared with those of WT/WT(Y359V) ϭ 3:1. There is no significant difference between the two groups (n ϭ 4 in WT/WT(Y359V) and n ϭ 3 in ⌬e2/WT(Y359V), p Ͼ 0.05). The data of WT/WT(Y359V) group here are the same as presented in C.
indicates the efficiency of trafficking to PM, was ϳ0.8 in cells expressing BK␣WT. In contrast, this ratio was almost zero in cells expressing BK␣⌬e2 (Fig. 4B).
When BK␣WT transcripts labeled with either YFP or CFP were co-expressed in HEK293 cells, the WT-CFP/WT-YFP complex demonstrated distinct and significant co-localization along the PM (Fig. 4C). In contrast, when WT-CFP and ⌬e2-YFP were co-expressed, both the fraction of WT-CFP in PM versus cytosol (Fig. 4D) and the ratio of WT-CFP co-localized with ⌬e2-YFP in the PM (Fig. 4E) were much smaller than those in WT-CFP and WT-YFP co-expression, respectively. Moreover, WT-CFP was highly co-localized with BK␣⌬e2 only in the In merged and expanded areas (enclosed by squares) in WTϩWT, significant co-localization (yellow) at the PM is indicated by an arrow. In WTϩ⌬e2, CFP fluorescence (red) rather than co-localization at the PM is indicated by an arrowhead. Cells were transfected with cDNA at a ratio (CFP:YFP) of 1:1. D, the ratio of fluorescence intensity of WT-CFP in PM to that of cytosol region. **, p Ͻ 0.01. E, co-localization ratio of WT-CFP in WT-CFPϩWT-YFP (black column) or WT-CFPϩ⌬e2-YFP (white) cells was analyzed in PM and Cytosol. **, p Ͻ 0.01 versus WTϩWT in PM. ##, p Ͻ 0.01 versus WTϩ⌬e2 in cytosol. F, BiFC assay was performed to detect direct coupling between WT and ⌬e2. A representative set of images from three independent experiments is presented. Cells were transfected with cDNA at a ratio (WT-VC155:⌬e2-VN173) of 1:1. G, FRET analysis based on acceptor photobleaching was carried out using WT-YFP, ⌬e2-YFP, and WT-CFP. Cells were transfected with cDNA at a ratio (CFP:YFP) of 1:1. **, p Ͻ 0.01 versus WT-CFP alone. H, co-IP assay was performed using HEK293 cells expressing WT-GFP ϩ FLAG-WT-mCh, WT-GFP ϩ FLAG-⌬e2-mCh, or only WT-GFP (for a negative control). Lysates were precipitated with anti-FLAG M2 antibody and blotted using the anti-GFP antibody. Similar results were obtained from three independent experiments. Cells were transfected with cDNA at a ratio (GFP:mCh) of 1:1. cytosol (Fig. 4E, p Ͻ 0.01 versus WTϩWT in PM and WTϩ⌬e2 in cytosol). Thus, hetero-tetramer formation of BK␣⌬e2 with BK␣WT reduces the trafficking efficiency of these complexes to the PM.
Heteromeric BK␣ formation was also examined using a bimolecular fluorescence complementation (BiFC) assay (10) (Fig. 4F). As shown in Fig. 4F, Venus fluorescence was consistently detected in HEK293 cells that co-expressed BK␣⌬e2 and BK␣WT tagged with N (VN173) and C (VC155) termini of Venus, respectively. To examine further the possible interactions between BK␣WT and BK␣⌬e2, FRET analysis based on an acceptor photobleaching method was also carried out ( Fig.  4G) (15,16). FRET was observed consistently in cells that coexpressed WT-YFP and ⌬e2-CFP. The results from this coimmunoprecipitation (co-IP) assay also demonstrate molecular interaction between BK␣⌬e2 and BK␣WT in HEK293 cells (Fig. 4H). In summary, our analysis shows that BK␣⌬e2 binds to both BK␣WT and BK␣⌬e2 with similar affinity to form heteromeric molecular complexes. Importantly, however, heterotetramer formation of BK␣WT with BK␣⌬e2 reduced the trafficking efficiency.

Molecular Imaging Reveals Stoichiometry of the BK␣WT/ BK␣⌬e2
Hetero-tetramer-The stoichiometry of BK␣WT/ BK␣⌬e2 heteromeric channels was further examined by employing single molecule imaging methods based on TIRF microscopy (15,17). Under TIRF illumination, the expression of only WT-mCh transcripts resulted in a distinct dotted pattern of signals (Fig. 5A). In contrast, expression of ⌬e2-GFP alone showed very weak fluorescence signals in the TIRF field, and it exhibited bright fluorescence under epifluorescence microscopy, suggesting mainly intracellular localization. When WT-mCh and ⌬e2-GFP were co-expressed, some of ⌬e2-GFP particles could be detected in the PM, where they were co-localized with WT-mCh particles. The combined signals due to mCh and GFP, detected as yellow particles, progressively moved together on the PM during 60-s measurements (Fig. 5A and see also the supplemental movie). This strongly suggests specific hetero-tetramer formation at or very near the PM.
The stoichiometry of these hetero-tetramers was also studied using molecular imaging, which quantified the "step down" decrease in fluorescent intensity (a single molecule GFP bleaching method) (15,17). In this approach: two types of cells co-ex- Here WT-GFP spots detected alone (not with WT-mCh) were analyzed. Note that the distribution is similar to theoretical frequency distribution assuming (i) WT-GFP homo-tetramers (n ϭ 4) and (ii) the apparent probability of GFP being fluorescent (p) as 0.8 (fit for tetramer). D, frequency distributions of the number of bleaching steps in co-localizing WT-GFP (red) or ⌬e2-GFP (blue) (50 and 46 particles from 5 and 6 cells, respectively). Cells were transfected with cDNA at a ratio (GFP:mCh) of 1:1. Note that in both cases the distributions show the peaks at 2, and these were well fitted to the binomial distribution with n ϭ 4 and p ϭ 0.5.
pressing WT-mCh and WT-GFP or expressing WT-mCh and ⌬e2-GFP were prepared by transfection of cDNA at a ratio of 1:1. Single spots of WT-GFP, which were detected alone (not with WT-mCh) in cells co-expressing WT-mCh and WT-GFP, displayed mainly 3 or 4 step bleaching (Fig. 5C). The population distribution of the data obtained from this bleaching step analysis was well fitted to the theoretical binomial distribution for a tetramer (n ϭ 4), with the apparent probability of GFP being fluorescent during excitation (p) set at 0.80 (18) (see "Experimental Procedures"). This result provided evidence that each GFP spot contained single BK␣WT tetramer under these conditions. In the secondary analysis, the pattern of bleaching of the GFP spots, which were detected by co-localization of mCh and GFP as yellow signals and assumed to represent heteromers of WT-mCh and ⌬e2-GFP (circles in Fig. 5B), was counted. Although these spots exhibited bleaching steps corresponding to one to four subunits, the population distribution peaked at two-step bleaching (Fig. 5D). Similarly, the presumed heteromer of WT-GFP and WT-mCh yielded a distribution that peaked at two steps. Both distributions were well fitted by binomial distribution, given n ϭ 4 and apparent fluorescence probability p ϭ 0.5. Here, p ϭ 0.5 is based on the premise that two molecules (A and B) possess the same affinity between A-A, A-B, and B-B, when composing homo-or hetero-tetramer. It is notable that both distributions of WT-GFP/WT-mCh and ⌬e2-GFP/WT-mCh are peaked at two-step bleaching and well fitted to the theoretical distribution. These results suggest that binding affinity between ⌬e2-GFP and WT-mCh is comparable with that between WT-GFP and WT-mCh and support the finding by FRET shown in Fig. 4G. Taken together, BK␣⌬e2 can form tetramers with BK␣WT at the stoichiometry of 1:3, 2:2, and 3:1.
The Second Short ␣-Helix Region in S0-S1 Linker (Helix2) Is Responsible for Both the Cell Surface Expression and the Voltage Dependency-The region in exon2 that is responsible for surface expression was examined using site-directed mutagenesis ( Fig. 6A and supplemental Table S4). Channel function and surface expression were measured based on whole-cell patch clamp recordings and anti-FLAG antibody imaging, respectively (see Fig. 2, A and B, Fig. 4, A and B, and supplemental Table S4). Mutants of ⌬127-140 (⌬1) and ⌬141-150 (⌬2) resulted in BK channel activity that were very similar to BK␣WT. Mutations of ⌬151-161 (⌬3) showed smaller but significant surface expression and only very little BK channel activity (n ϭ 5). Other variants (⌬162-170 (⌬4), ⌬171-178 (⌬5), or ⌬179 -180 (⌬6)) totally failed to generate BK currents and cell surface expression (Fig. 6B).
To examine this site-specific regulation further, eight sets of mutants were prepared by substitution of two or three sequential amino acids with alanine in the sequence from 159 to 180 (MTS, VKD, WAG, VMI, SAQ, TLT, GR, and VL in Fig. 6A). The variants of MTS, VKD, WAG, VMI, and VL showed significantly smaller whole-cell BK current. However, these mutants localized to the PM to a similar extent as BK␣WT (Fig. 6C). Voltage dependence of these mutants was obtained from the activation curves at pCa 6.5 and compared by the half-activation voltage (V 1/2 ) (see "Experimental Procedures"). As shown in Fig. 6E, the activation curves of ⌬3, MTS, VKD, WAG, VMI, and VL showed substantial rightward shift in comparison with WT. Accordingly, V 1/2 values were elevated ( Fig. 6F and supplemental Table S5). For example, VMI showed PM expression comparable to WT but almost no current at ϩ120 mV (Fig. 6C). The lack of current in VMI may be attributable to the marked shift of V 1/2 to positive direction by ϩ150 mV (Fig. 6F). As for the variant of TLT, whole-cell BK channel currents at pCa 6.5 were significantly increased. However, V 1/2 at pCa 6.5 was similar to that of WT. All mutants in Fig. 6, E and F, possess significant Ca 2ϩ sensitivity (supplemental Fig. S3 and Table S5).
On the other hand, SAQ and GR mutants exhibited neither surface expression nor BK currents (Fig. 6C). When Ser 171 or Gln 173 was substituted by alanine (S171 and Q173 in Fig. 6D, respectively), the PM expression was not altered, whereas the average current density significantly decreased (Fig. 6D). It is apparently due to the shift of V 1/2 to depolarized potentials (Fig.  6, E and F). The Ala substitution of Gly 177 or Arg 178 (G177 and R178 in Fig. 6D, respectively) decreased both BK current and surface expression. In fact, Arg 178 is a key amino acid for trafficking. Taken together, amino acids in the S0-S1 linker, especially the Helix2 (especially Ser 171 and Gln 173 ) and Helix2-S1 linker (Gly 177 and Arg 178 ), are necessary for both BK␣ trafficking to PM and channel function, including voltage dependence.
Additional experiments further documented the functional significance of the splice variant. It is well known that histamine can enhance physiological responses in chondrocytes, such as increasing prostaglandin E (PGE) production (20). Accordingly, the effect of the same siRNAs that altered channel function was assessed in terms of the expression of COX2, an inducible enzyme for PGE 2 production (Fig. 7F). Measurements based on Q-PCR showed that COX2 expression in OUMS-27 pretreated with 30 M histamine for 24 h was ϳ2ϫ higher than that in untreated cells (siCTR normalized to untreated cells: 1.9 Ϯ 0.2, n ϭ 5). Importantly, siWT slightly decreased (siWT: 1.3 Ϯ 0.1, n ϭ 5), and si⌬e2 significantly increased COX2 mRNA expression (si⌬e2: 2.7 Ϯ 0.3, n ϭ 5).

Discussion
Main Findings-A new splice variant of BK channel ␣ subunit (BK␣⌬e2) was cloned from OUMS-27, an accepted model of human chondrocytes. Our results suggest that this transcript has a significant role in articular joint function. It may also be the case under pathophysiological conditions, as this transcript is found in chondrocytes from osteoarthritis patients as well (Fig. 1C).
Homo-tetrameric BK␣⌬e2 transcripts cannot traffic to PM when transfected into HEK293 cells (Fig. 4, A and B). Electrophysiological (Figs. 2 and 3) and imaging (Figs. 4 and 5) analyses show that BK␣⌬e2 can form hetero-tetramers with BK␣WT and are functionally expressed in PM. The affinity between BK␣WT and BK␣⌬e2 for heteromerization is similar to that between BK␣WT subunits based on FRET and GFP bleaching data (Figs. 4G and 5D). We note that hetero-tetramer complexes consisting of WT/⌬e2 (at a ratio of 3:1, 2:2, or 1:3) can traffic to the PM, where functional BK channels are formed. Overall, the hetero-tetramerization of BK␣⌬e2 with BK␣WT reduces both the efficiency of BK␣WT trafficking to PM (Fig. 4) and the single channel functions (Figs. 2, 3, and 6). These effects occur in a stoichiometry-dependent manner.  Table S4. *, p Ͻ 0.05; **, p Ͻ 0.01 versus WT by Dunnett's test. E, voltage dependence was examined in mutants that showed substantial reduction of current density without marked reduction of membrane trafficking, i.e. ⌬3, MTS, VKD, WAG, VMI, TLT, VL, S171 and Q173. Intracellular Ca 2ϩ level was fixed at pCa 6.5. Relationships between G/G max and voltage were fitted with Boltzmann equation. F, the values of half activation voltage (V 1/2 ) of mutants were compared. *, p Ͻ 0.05; **, p Ͻ 0.01 versus WT.
Our analyses based on deletion mutants in exon2 (⌬1-⌬6) revealed that Helix 2 (especially Ser 171 and Gln 173 ), the adjacent linker to the S1 segment (Gly 177 and Arg 178 ), and a part of S1 segment are essential for the surface expression (Fig. 6). A short ␣-helix in the C terminus of BK␣WT ( 1112 DLIFCL 1117 ) is known to function as an ER export motif (21). Indeed, intracellular BK␣⌬e2 was substantially co-localized with an ER marker (data not shown). Therefore, the Helix2 in S0-S1 linker may be involved in the mechanism for surface trafficking of BK channel via the interaction with a cargo recognition component (22) or an adaptor protein complex (23).
The deletion of exon2 affected not only the trafficking efficiency but also channel kinetics when it forms hetero-tetramers with BK␣WT (Fig. 2, F and G). Deletion mutant (⌬3: ⌬151-161) and mutants of alanine replacement (MTS, VKD, WAG, VMI, VL, Ser 171 and Gln 173 ) exhibited smaller BK current density even though their surface expression was comparable with that for BK␣WT (Fig. 6, B-D). Analyses of voltage dependence revealed that voltage sensitivity of these mutants was markedly reduced (Fig. 6, E and F). It has been reported that Asp 164 in the 162 VKD 164 forms a Mg 2ϩ binding site, and Mg 2ϩ binding is essential for interaction between voltage-sensor domain and cytoplasmic domain (RCK1) (24). This may be the reason why the mutation of 162 VKD 164 exhibits impaired voltage sensitivity. 159 MTS 161 , 165 WAG 167 , 168 VMI 170 , Ser 171 , Gln 173 , and 179 VL 180 are located closely to Asp 164 and, therefore, mutation intheseresiduesmayalsoaffectMg 2ϩ bindingandvoltagedependence. Such effects could explain the observed smaller single channel conductance and shorter mean open time of WT/⌬e2 BK channels (Fig. 2).
In this mutation assay some contradictions were observed. For example, deletion of 179 VL 180 (⌬6) prevents surface trafficking (Fig. 6B), whereas alanine replacement of 179 VL 180 (VL) does not affect surface trafficking (Fig. 6C). We assume that deletion of 179 VL 180 causes larger changes in the conformation of Helix2, Helix2-S1 linker, and/or S1 segment than Ala substitution. This conformational change in ⌬6 may prevent Helix2 from binding to a cargo recognition protein. A similar explanation would be possible for discrepancy between ⌬162-170 (⌬4) and mutants of Ala replacement in the region of Val 162 -Ile 170 .
When [Ca 2ϩ ] i was increased to 10 M (pCa 5.0, see supplemental Fig. S3 and Table S5), V 1/2 of all mutants in Fig. 6, E and F, significantly decreased, whereas the sensitivity to Ca 2ϩ was different among the mutants. Thus, Helix2 and adjacent regions may partially contribute to Ca 2ϩ sensitivity (25).
Finally, the effects of single mutations of Ser 171 , Gln 173 , Gly 177 , and Arg 178 should be emphasized. Both Ser 171 and Gln 173 exhibited a striking shift of V 1/2 over ϩ75 mV without the significant change in trafficking efficiency observed in SAQ. In contrast, Gly 177 and Arg 178 exhibited both reduced wholecell BK channel currents and impaired trafficking efficiency. Arg 178 may be a key amino acid in the linker between Helix2 and S1 segment to keep the Helix2 in the correct position or angle required for trafficking efficiency and channel activity. Taken together, it can be concluded that BK␣⌬e2 strongly inhibits BK channel function by two mechanisms; (i) preventing surface expression (Figs. 4 and 6) and (ii) reducing single channel activity by decreasing single channel conductance (Fig.  2) and voltage-sensitivity (Fig. 6).
Translational Significance of BK Channel Complexes-Inflammation in synovia of articular joints leads to an increase in mast cell migration, and these cells release pro-inflammatory mediators such as histamine, PGE 2 , IL-1, and TNF-␣. Downstream actions of these agents can initiate the progressive degenerative diseases such as OA (26). In healthy articular cartilage, histamine binds to its type-1 receptor (H 1 R) and promotes cell proliferation. This is in part due to an increase in [Ca 2ϩ ] i and related changes in DAG and in PKC activities. Notably, in cartilage from OA patients, expression levels of H 1 R and histidine decarboxylase are higher than normal (26). These changes may enhance H 1 R signaling and lead to increased PGE 2 and metalloproteinase-3 (MMP-3) and -13 production (20) as well as cell proliferation (26). Histaminemediated signaling pathway is involved in the inhibition of matrix production and resulting degeneration of articular cartilage under OA pathogenesis.
In the present study histamine promoted gene expression of COX2. Furthermore, the BK␣⌬e2 knockdown resulted in an increase in both BK channel current density and COX2 transcripts (Fig. 7). The COX2 transcription is regulated by several Ca 2ϩ -dependent factors (27). Overall our results suggest that an increase in [Ca 2ϩ ] i can activate nuclear factor of activated T-cells (NFAT) and/or nuclear factor B (NFB), leading to COX2 transcription. Previously, we demonstrated in OUMS-27 cells that K Ca channels (including the BK, intermediate and small conductance Ca 2ϩ -activated K ϩ (IK and SK) channels) are functionally expressed. Moreover, BK channels are essential for enhancement of histamine-induced Ca 2ϩ influx, as the BK channel-mediated hyperpolarization of the resting membrane potential enhances Ca 2ϩ influx (8). Here, we have shown that the BK␣⌬e2 knockdown can cause up-regulation of BK channel activity and membrane hyperpolarization and subsequently enhanced Ca 2ϩ influx. This increase in [Ca 2ϩ ] i may activate NFAT and/or NFB and, thus, promote COX2 transcription.
The relative mRNA ratio of BK␣⌬e2 to BK␣WT (WT:⌬e2) was 1:0.14 in OUMS-27 cells (Fig. 1D). Experiments using HEK293 heterologous system actually showed that 1 ⁄ 10 the amount of BK␣⌬e2 plasmid (1:0.1) reduced whole-cell BK channel currents by approximately 40% (supplemental Fig. S2). Moreover, co-transfection of BK␣⌬e2 cDNA inhibited wholecell BK channel currents in a ratio-dependent manner. When mRNA ratio (WT:⌬e2) is 1:0.14, the probability that BK␣ tetramer contains one or more BK␣⌬e2 subunits is calculated as approximately 41%. When si⌬e2 reduces the BK␣⌬e2 mRNA level to 60% in OUMS-27, i.e. WT:⌬e2 is 1:0.084, the probability that a BK␣ tetramer contains BK␣⌬e2 subunit(s) is approximately 28%. Thus, treatment with si⌬e2 is thought to increase the occurrence of BK␣WT homo-tetramers by 13% even if the increase in trafficking efficiency is not taken into account. In addition, the stoichiometric change from 1⌬e2: 3WT to 4WT increases the single channel conductance by ϳ20% (Fig. 2). All these factors are considered to contribute to significant enhancement of whole-cell BK channel current amplitude. Thus, it is rather reasonable that siRNA knockdown of ⌬e2 substantially increases whole-cell BK channel current and COX-2 induction.
The expression level of BK␣ is up-regulated in OA patients (12). Similarly, immunohistochemical analysis demonstrated that BK␣ is up-regulated in the middle region of articular cartilage from OA animal models (28). We observed that inflammatory factors, such as IL-1␤ and TNF-␣, modulate the mRNA levels of BK␣⌬e2 in OUMS-27 cells (data not shown). Thus, it is possible that changes in the expression of BK␣⌬e2 versus that of BK␣WT (specifically, the regulation of the splicing) may be involved in the pathogenesis and/or progression of OA. The expressional changes in BK␣WT and/or BK␣⌬e2 in chondrocytes of OA patients and the relevance of them to pathogenesis should be studied in future. An increase in [Ca 2ϩ ] i is involved in development of OA by activating calcium/calmodulin-dependent protein kinase II (29). Accordingly, the ion channels that modulate [Ca 2ϩ ] i changes are critically important for the treatment of OA.
In summary, BK␣⌬e2, a newly identified splice variant of BK channels can significantly regulate human chondrocyte function under physiological and/or pathophysiological conditions. It does so by serving as a negative factor for trafficking to PM after formation of hetero-tetramers with BK␣WT and also by regulating channel activity.

Experimental Procedures
Cell Culture, Human Tissue, and Animal Sources-HEK (human embryonic kidney) 293 and OUMS-27, the human chondrocyte cell line, were supplied from Japanese Collection of Research Bioresources Cell Bank and cultured as described previously (8 -10). C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). Human articular cartilage from (i) a healthy individual and (ii) a patient with osteoarthritis was purchased from Articular Engineering (Northbrook, IL). All experiments were approved by the Ethics Committee of Nagoya City University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.
Electrophysiological Recordings-Electrophysiological studies were performed as described previously (16). When making whole-cell BK current measurements, the pipette solution contained 140 mM KCl, 2.8 mM MgCl 2 , 10 mM HEPES, 2 mM Na 2 ATP, 5 mM EGTA, and 3.15 mM CaCl 2 (pCa 6.5). When the effect of increase in intracellular Ca 2ϩ on mutants was examined, 4.93 mM CaCl 2 was added to fix the intracellular Ca 2ϩ concentration at 10 M (pCa 5.0). The pH was adjusted to 7.2 with KOH. The extracellular solution had an ionic composition 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl 2 , 1.2 mM MgCl 2 , 14 mM glucose, and 10 mM HEPES. The pH was adjusted to 7.4 with NaOH. Whole-cell BK currents were activated from a holding potential of Ϫ80 mV by applying 150-ms voltage steps, once every 10 s, to a voltage range between Ϫ60 and ϩ120 or ϩ140 mV in increments of 20 mV. The activation curve was made by plotting relative tail current amplitudes (G/G max ) recorded at Ϫ60 or Ϫ30 mV against test potential ranging from Ϫ100 to ϩ200 mV or from Ϫ60 mV to ϩ260 mV. Obtained data were fitted with a Boltzmann relationship, G/G max ϭ (1 ϩ exp((V Ϫ V 1/2 )/S)) Ϫ1 , where V 1/2 , V, and S are the voltages required for half-maximum activation, membrane potential, and slope factor, respectively. Data were sampled at 10 kHz and filtered at 2 kHz. The BK channel-mediated current was defined as the 1 M paxilline-sensitive current (15). The resistance of the pipette was 2-5 megaohms for whole-cell patch clamp configurations when filled with the pipette solutions.
Single BK channel current recordings were made using the inside-out patch clamp method. The compositions of solutions were as follows: pipette solution: (A) control measurements: 140 mM KCl, 1.2 mM MgCl 2 , 1.82 mM CaCl 2 , 5 mM EGTA (pCa 7.0), 14 mM D-glucose and 10 mM HEPES; (B) TEA-insensitive current recordings: 140 mM KCl, 1.2 mM MgCl 2 , 2.2 mM CaCl 2 , 5 mM EGTA (pCa 6.8), 14 mM D-glucose, 10 mM HEPES, and 2 mM TEA-Cl (pH 7.2 with KOH); bath solution: 140 mM KCl, 1.2 mM MgCl 2 , 3.15 mM CaCl 2 , 5 mM EGTA (pCa 6.5), 14 mM D-glucose, and 10 mM HEPES (pH 7.2 with KOH). All single-channel data were sampled at 5 kHz, filtered at 0.5 kHz, and recorded for 60 s after channel activity was stable. The data acquisition protocol was repeated at least three times for each preparation. All patches were voltage-clamped to 0 mV, and their unitary currents were activated by voltage steps to 50 mV. For single channel amplitude analysis, only patches containing fewer than two channel types were utilized. The unitary current amplitudes were obtained by fitting data with the Gaussian function. Single