Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M704440200 on November 8, 2007

J. Biol. Chem., Vol. 283, Issue 5, 2709-2715, February 1, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/5/2709    most recent
M704440200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lv, C.
Right arrow Articles by Ding, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lv, C.
Right arrow Articles by Ding, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Four-turn {alpha}-Helical Segment Prevents Surface Expression of the Auxiliary hβ2 Subunit of BK-type Channel*

Caixia Lv{ddagger}1, Maorong Chen{ddagger}1, Geliang Gan{ddagger}1, Lifen Wang{ddagger}, Tao Xu{ddagger}§2, and Jiuping Ding{ddagger}3

From the {ddagger}Key Laboratory of Molecular Biophysics (Huazhong University of Science and Technology), Ministry of Education, Wuhan, Hubei 430074, China and the §National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China

Received for publication, May 30, 2007 , and in revised form, October 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Large conductance, voltage- and Ca2+-activated K+ (BK) channels encoded by the mslo {alpha} and β2 subunits exist abundantly in rat chromaffin cells, pancreatic β cells, and DRG neurons. The extracellular loop of hβ2 acting as the channel regulator influences the rectification and toxin sensitivity of BK channels, and the inactivation domain at its N terminus induces rapid inactivation. However, the regulatory mechanism, especially the trafficking mechanism of hβ2, is still unknown. With the help of immunofluorescence and patch clamp techniques, we determine that the hβ2 subunit alone resides in the endoplasmic reticulum, suggesting that trafficking mechanism of hβ2 differs from that of hβ1 opposite to what we predicted previously. We further demonstrate that a four-turn {alpha} helical segment at the N terminus of hβ2 prevents the surface expression of hβ2, that is, the helical segment itself is a retention signal. Using the c-Myc epitope-tagged extracellular loop of hβ2, we reveal that the most accessible site by antibody is located at the middle of the extracellular loop, which might provide clues to understand how the auxiliary β subunits regulates the toxin sensitivity and the rectification of BK-type channels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Large conductance voltage- and Ca2+-activated K+ (BK)4 channels associated with its auxiliary β subunits show functional varieties in many native cells (1–13). The auxiliary β1–β4 subunits of BK channels are composed of two transmembrane segments and a large extracellular segment (5, 14–16). For hβ2 subunits, three hydrophobic residues FIW at its N terminus produces a rapid inactivation of BK channels (15), whereas it does not follow a typical N-type inactivation mechanism (17). The NMR experiment reveals a four-turn helix structure located at the middle of its N terminus (24). Recently, Zhang et al. (18) reported that trypsin can easily target the Arg8 and Arg9 of nine basic residues before the helix. In addition, its extracellular loop prevents the scorpion toxin charybdotoxin from approaching the channel pore (9) and induces the rectification of BK channels (19). It has been reported that the β1 and β2 subunits contain endocytic sorting signals at their C termini, which can down-regulate the association MaxiK channel surface expression levels (14, 20). The β1 subunit can reach the cell membrane alone, whereas the β2 subunit only appears underneath the plasma membrane (14, 20, 21). Obviously, the trafficking mechanism of hβ2 subunits is still unknown so far.

There are many mechanisms for governing the surface expression of ion channels and thus the electrical activity of a cell. A channel protein in a misfolded or unfolded state can be trapped in the ER as demonstrated by measurements of the surface labeling, luminomitry. Some retrieval/retention and anterograde signals determine the ER exit of proteins (22). The retention sequence RKR appearing in potassium inward rectifying channel (Kir) and its auxiliary SUR1 subunit is due to improperly folded or assembled channels (22, 23). Both the Kir and SUR1 subunits have the cytosolic RKR sequences, which must be masked by the assembly of octamer before channels can be transported to the cell surface (23). Zarei et al. (24) also report that a nonbasic hydrophobic retention/retrieval motif CVLF prevents the surface expression of mslo {alpha} and β1-subunits.

With the help of immunofluorescence and patch clamp techniques, we find that the auxiliary hβ2 subunit cannot traffic to the cell surface without associating with the mslo {alpha} subunit. Fortunately, the hβ2 subunit can solely traffic to the cell surface only after demolishing or destroying a four-turn {alpha} helical segment of its N terminus (25), which may implicate a novel trafficking mechanism. We also find that the most accessible region by antibody is located at the middle position of the hβ2 outer loop near the channel filter, suggesting that it may be the important site in regulating the toxin sensitivity and rectification of inactivating BK currents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Constructions and Mutagenesis—The full-length cDNA for each of the mslo {alpha} or hβ2 were subcloned into pcDNA3.1/Zeo(+) (Invitrogen). For the construct hβ2-Tdimer2, hβ2 cDNA, and Tdimer2 cDNA were subcloned into pcDNA3.1/Zeo(+). The BK pore-forming {alpha} subunit (mslo) was tagged with the c-Myc epitope (EQKLISEEDL) at the N terminus. To get a green fluorescence protein targeted ER, pEGFP-ER, the enhanced green fluorescence protein (EGFP) was used to substitute the DsRed2 in pDsRed2-ER (Clontech). The human β subunits (hβ1/hβ2) were tagged with epitope (c-Myc, EQKLISEEDL; hemagglutinin, YPYDVPDYA) at different positions of the putative extracellular loop by sequential overlap extension PCR. Series of constructs for deletions of the hβ2–137myc N terminus were made by deleting appropriate amino acids from hβ2–137myc and introducing a new start codon with PCR primers. The mutations on hβ2–137myc were created with the QuikChange site-directed mutagenesis kit (Stratagene). All of the constructs and mutations were verified by direct DNA sequence analysis.

Immunofluorescence Imaging in HEK293 Cells—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin. The cells were transiently transfected using Lipofectamine 2000 (Invitrogen). After transfection, the live cells were transferred to a poly-D-lysine-coated chamber. The next day, the cells were fixed with 2% paraformaldehyde in phosphate-buffered saline for 5 min. For cell permeabilization, 0.2% Triton X-100 was added for 5 min. After blocked with 5% goat serum for 1 h, the cells were incubated with a monoclonal anti-human c-Myc antibody (1:200) for 3 h, washed, and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (H+L) (1:150) for 1 h. All of the experiments were performed at room temperature (22–25 °C). Mouse anti-human c-Myc and fluorescein isothiocyanate rabbit anti-mouse IgG (H+L) conjugate were purchased from Zymed Laboratories Inc.

We took advantage of the high numeric aperture objective (APO x100 OHR, NA = 1.65, Olympus) to take high resolution fluorescence images of transfected cells. Excitation light from a fiber optical coupled monochromator (Polychrome IV; TILL Photonics GmbH, Germany) was passed through a shutter that opened only during camera exposure. The wavelength selection and switch were controlled by the image acquiring software (TILL vision 4.0; Till Photonics GmbH). The images were acquired with a cooled CCD (PCO SensiCam; Germany) with pixel size of 0.067 µm at the specimen plane.

Image Analysis and Statistics—Images were viewed, processed, and analyzed in TILL Vision (TILL.Photonics, Germany) and Adobe Photoshop (Adobe Systems) and IMAGE J (National Institutes of Health, Public Domain). The exposure time was 2000 ms. Control experiments were executed for each batch of experiments. All of the experiments were performed at there different batches. At each batch, every chamber is normalized to the control.

Solutions—HEK293 cells were bathed in ND-96 solution (pH 7.5) containing the following: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2.5 mM C3H3O3Na (sodium pyruvate), and 10 mM HEPES. For current recording of BK channels, extracellular solution contained the following: 160 mM MeSO3K, 2 mM MgCl2, 10 mM HEPES, pH 7.0, titrated with MeSO3H; intracellular solution contained the following: 160 mM MeSO3K, 10 mM HEPES, 5 mM N-hydroxyethylenediaminetriacetic acid with added Ca2+ for 10 µM free Ca2+ and 5 mM EGTA with no added Ca2+ for 0 µM Ca2+ solution, as defined by the EGTAETC program (McCleskey, Vollum Institute, Portland, OR), with the pH adjusted to 7.0, titrated by MeSO3H. All of the chemicals were attained from Sigma.

Electrophysiology—Patch pipettes were pulled from borosilicate glass capillaries with a resistance of 2–4 megohms when filled with pipette solution. Inside-out patches were used in experiments. During recordings, 0 and 10 µM Ca2+ solutions were applied onto the cell via a perfusion pipette containing seven solution channels. All of these experiments were performed at room temperature (22–25 °C). The experiments were performed using a PC2C patch clamp amplifier with its software (InBio). The currents were typically digitized at 100 kHz. Macroscopic records were filtered at 5 kHz. Patch clamp recording data were analyzed with IGOR (Wavemetrics, Lake Oswego, OR), Clampfit (Axon Instruments, Inc.), and Sigmaplot (SPSS, Inc.) software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Auxiliary hβ2 Subunits Alone Reside in the ER—The hβ subunits of a four-member β family (hβ1–hβ4) have the similar structure, i.e. with two transmembrane segments, an extra large loop, and a few consensus glycosylation sites (26) (Fig. 1A). The hβ2 subunits like other β subunits have many ways to regulate the mslo BK channels. For example, its extracellular loop alters the conductance and toxin sensitivity of BK channels, but its N terminus introduces a rapid inactivation of BK channels (9, 17, 19). The hβ1 subunits have been reported and can traffic to the cell surface alone (14). However, it is unclear whether the auxiliary hβ2 subunits also traffic to the plasma membrane as hβ1 subunits do. To investigate the trafficking mechanism of auxiliary hβ2 subunits, a c-Myc epitope was tagged to the center of the loop for measuring the surface expression of hβ2 subunits (Fig. 1A). Considering that recognition signals (export and retention/retrieval) of proteins in the ER are mostly found at the N or C termini, we constructed a series of mutations at the N terminus to investigate the trafficking mechanism of the hβ2 subunits (Fig. 1B). A three-dimensional structure of the N-terminal helix from 18 to 31 is plotted in the Fig. 1C (25).

Because coexpression of mslo {alpha} and hβ2 subunits can make the inactivating functional BK channels, we expected that images for coexpression of Myc-mslo (green) and hβ2-Tdimer2 (red) should show their coexistence in HEK293 cells. In fact, images for Myc-mslo, hβ2-Tdimer2, and Myc-mslo+hβ2-Tdimer2 show green (left), red (middle), and yellow (right) brims, respectively, which indicates coexistence of two proteins on the cell surface (Fig. 2A). In contrast, there was no red contour distinctly appearing on the cell brim, whereas the hβ2-Tdimer2 was transfected alone in HEK293 cells. The image of coexpression of pEGFP-ER with hβ2-Tdimer2 demonstrates that two proteins coexist in the ER (Fig. 2B). Furthermore, there was no detectable colocalization with coexpression of the hβ2-Tdimer2 with an EGFP-tagged Golgi marker (data not shown) that meant the hβ2 subunit should be retained in ER. Because the Tdimer2 is a protein with more than 450 amino acids, it may influence the traffic of hβ2. To precisely recognize and quantify surface expression of hβ2 subunits, a c-Myc epitope was tagged at the loop. Similarly, the image for coexpression of mslo with hβ2–137myc revealed that the hβ2–137myc subunits were transported to the membrane surface (Fig. 2C, right panel). It is interesting that the surface signals (green) of the hβ2–137myc subunits alone could be detected only under permeabilized conditions (Fig. 2C, left and middle panels). In other words, the green signals were undetectable under nonpermeabilized conditions, whereas the hβ2–137myc subunits were transfected alone in HEK293 cells (Fig. 2C, left panel). In conclusion, a surprising finding is that the hβ2 subunit cannot traffic to the plasma membrane alone except that it expresses with the mslo {alpha} subunits together.


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 1.
Mutagenesis at the N terminus and extracellular loop of the auxiliary hβ2 subunits of BK channels. A, topology of hβ2 subunits exhibits a putative structure, which consists of a N terminus, two transmembrane domains (TM1 and TM2), a long extracellular loop, and a short C terminus. The N terminus contains three components: an inactivation "ball" domain, am {alpha}-helical segment (dark gray), and two flexible linkers. Four conserved cysteines (medium-dark gray) in the extracellular loop are located to positions 84, 113, 142, and 174, respectively. In most experiments, ac-Myc epitope (light gray) was inserted at position 137 of the extracellular loop. B, a series of N-terminal mutations and constructs of hβ2 subunits is shown in the list. The residues 18–31 of the{alpha} helical segment are labeled in light gray, and the substituting residues are in dark gray. C, a three-dimensional structure of the four-turn helix from 18 to 31 of hβ2 subunits gives more detailed orientation of residues. The hydrophobic residue is labeled in light gray, the negatively charged one is in dark gray, and the positively charged one is in medium-dark gray.

 


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 2.
The hβ2 subunits are restricted to the ER. A, HEK293 cells were co-transfected with Myc-mslo (green) and hβ2-Tdimer2 (red) subunits. Images for Myc-mslo (left panel) and hβ2-Tdimer2 (middle panel) have clearly green and red brims, respectively, and the merged image (right panel) has a yellow brim, which demonstrated a colocalization of two proteins. The scale bar indicates 5 µm. B, HEK293 cells were co-transfected with pEGFP-ER (green) and hβ2-Tdimer2 subunits (red). The images for coexpression of pEGFP-ER and hβ2-Tdimer2 subunits demonstrated coexistence of two proteins in ER. The images are representative of 96 (top panels); 16, 54, and 53 (middle panels); and 81 (bottom panel) cells, respectively. The scale bar indicates 5 µm. C, HEK293 cells were transfected with hβ2–137myc (green) subunits under nonpermeabilized conditions, hβ2–137myc subunits with adding Triton under permeabilized conditions, and the mslo+hβ2–137myc subunits, which are shown in the left, middle, and right panels, respectively (see "Experimental Procedures"). The hβ2–137myc subunits alone do not appear at the cell surface under nonpermeabilized conditions (left panel) but clusters in ER under permeabilized conditions (middle panel). Coexpression of mslo+hβ2-myc137 subunits shows a hβ2 at the cell surface (right panel). The scale bar indicates 5 µm.

 
The hβ2 Subunit Is Restricted in the ER by Four-turn {alpha}-Helical Segment—To look for the retention signals of hβ2 subunits, we deleted the C terminus ({Delta}C) first and then the initial three hydrophobic residues FIW ({Delta}FIW). In Fig. 3A, mutations {Delta}C and {Delta}FIW failed to relieve hβ2 from being retained in ER, but they did traffic to the cell surface after coexpressing with the mslo subunits (data not shown). After deleting the first 10, 20, and 30 amino acids of N terminus, we found that the construct {Delta}30A was the only one appearing on the cell surface, which illuminates that the retention signals of the hβ2 subunits must be concealed among the residues ranging from 20 to 29. Examining their fractional intensities plotted in Fig. 3D, we further confirmed that only {Delta}30A could traffic to the cell surface.

The constructs {Delta}23A, {Delta}26A, and {Delta}30A were designed to precisely determine the retrieval signal or domain. After gradually decreasing the intensity of retention signals, we determined that the retrieval signal is a motif of 23–29 amino acids (Fig. 3B). To further determine the exact sequence of retention signals, the mutations {Delta}KIR and {Delta}DHDL were designed to relieve the retention signal of ER. In Fig. 3B, the construct {Delta}KIR is clearly a better mutation than the construct {Delta}DHDL for that purpose. It indicated that the motif KIR played a major role in trapping hβ2 in the ER, which is also consistent with the statistical data of the fractional intensities shown in Fig. 3E.

Because alanine-scanning mutations from Q23A to L30A did not show any remarkable surface expression (data not shown), we thus inferred that the motif KIR might be only a core of an expanded sequence. Considering that the motif KIR is located within a four-turn {alpha} helical segment ranging from residues 18 to 31 (25), we tested whether the whole helix served as a retention signal. A series of mutations, i.e. 23A–26A, 24A–26A, 24A–27A, and 23A–27A, were made to flatten the helix (Fig. 3C). Furthermore, mutant Q23P,D27P was constructed to destroy the helix by two prolines, because proline residue is usually thought to promote distortions of transmembrane helices (27, 28) (Fig. 3C). In Fig. 3 (E and F), the expressions of 23A–27A is about 30% more than that of 24A–26A or {Delta}KIR, which indicates that the motif KIR plays a role in trafficking through its secondary structure.


Figure 3
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 3.
Determination of detention motif of hβ2 at both the N and C termini by site-directed mutagenesis. A, images for HEK293 cells transfected with the constructs {Delta}C (a C-terminal deletion from Tyr221 to Arg235), {Delta}FIW, {Delta}10A, {Delta}20A, and {Delta}30A tagged with a c-Myc epitope at position 137 are shown. The scale bar indicates 5 µm. B, images for HEK293 cells transfected with the constructs and mutations {Delta}23A, {Delta}26A, {Delta}30A, {Delta}KIR, and {Delta}DHDL tagged with c-Myc epitope at position 137 are shown. The scale bar indicates 5 µm. C, images for HEK293 cells transfected with the mutations 24A–26A, 23A–26A, 24A–27A, 23A–27A, and {Delta}30A tagged with c-Myc epitope at position 137 are shown in the upper panels. The images for HEK293 cells co-transfected with Q23PD27P, hβ1. and QKIRD-hβ1 (a N-terminal mutation of hβ1) tagged a hemagglutinin epitope at position 98 are shown in the lower panels. The scale bar indicates 5 µm. D–G, the statistical value of fractional pixel intensity is plotted for each mutation as indicated. The fractional intensity of mutations was normalized to the controls mslo+{Delta}30A (D and E), {Delta}30A (G), and hβ1(3G), respectively (see "Experimental Procedures"). The percentage intensities of {Delta}C, mslo+{Delta}C, {Delta}FIW, mslo+{Delta}FIW, {Delta}10A, mslo+{Delta}10A, {Delta}20A, mslo+{Delta}20A, {Delta}30A, and mslo+{Delta}30A are 15.2 ± 1.8 (n = 21), 89.5 ± 10.9 (n = 36), 17.7 ± 3.0 (n = 16), 99.5 ± 8.8 (n = 58), 13.4 ± 1.9 (n = 14), 92.1 ± 11.4 (n = 43), 10.7 ± 0.5 (n = 20), 101.9 ± 5.5 (n = 74), 100.0 ± 7.2 (n = 75), and 100.0 ± 0.0 (n = 68), respectively (D). The percentage intensities of {Delta}23A, mslo+{Delta}23A, {Delta}26A, mslo+{Delta}26A, {Delta}30A, mslo+{Delta}30A, {Delta}KIR, mslo+{Delta}KIR, {Delta}DHDL, and mslo+{Delta}DHDL are 41.9 ± 4.5 (n = 23), 104.7 ± 10.8 (n = 62), 79.0 ± 10.1 (n = 97), 96.4 ± 7.7 (n = 38), 103.5 ± 11.9 (n = 59), 100.0 ± 0.0 (n = 84), 51.9±7.3 (n = 45), 94.3 ± 8.1 (n = 73), 15.8 ± 7.6 (n = 13), and 106.5 ± 10.0 (n = 41), respectively (D). The percentage intensities of 24A–26A, 23A–26A, 24A–27A, 23A–27A, {Delta}30A, and Q23PD27P are 51.9 ± 7.3 (n = 44), 68.8 ± 4.2 (n = 46), 68.1 ± 1.3 (n = 64), 84.2 ± 6.6 (n = 123), 100.0 ± 0.0 (n = 82), and 47.5 ± 9.2 (n = 46), respectively (F). The construct QKIRD-hβ1 is shown at the top inset in G. The substituting residues are labeled in gray. The percentage intensities of hβ1 and QKIRD-hβ1 are 100.0 ± 0.0 (n = 51) and 96.8 ± 1.6 (n = 44), respectively (G).

 
To further demonstrate whether the retention signal is a fixed sequence or a secondary structure, we inserted the QKIRD sequence into the N terminus of hβ1 subunits i.e. QKIRD-hβ1 (Fig. 3, C and G). The mutant QKIRD-hβ1 of hβ1 did not show any clear impact on the surface expression of hβ1 subunits. Therefore, the retention signal of hβ2 subunits is the four-turn helix itself rather than the primary sequence QKIRD. Furthermore, the motif KIR also appears in the βE of the C terminus of mslo subunits (29), which is obviously not a retention signal of BK channels.

To test whether the retention signal at the N terminus was able to present at other locations of hβ2, we translocated the potential retention signal, i.e. the first 33 amino acids of N terminus, to the C terminus of hβ2 after Arg235 (C33). The C33 alone shows little expression, whereas the mSlo+C33 only shows a little reduction compared with that of the wild type hβ2 (Fig. 4). It means that the same retention signal presents at the new location. We thus believe that the four-turn helix structure at the N terminus is a retention signal of hβ2.

Accessibility of the Extracellular Segment of hβ2 for Antibody Varies with Loci of c-Myc Epitope—We first define the c-Myc antibody accessibility at different positions in the outer segment/loop of hβ2. The differential fluorescence intensity of c-Myc antibody at positions in the hβ2 outer loop may arise from either the cysteine-rich and highly glycosylated structure of the loop or restrictions on access of the big antibody molecule to small volumes. The stronger fluorescence intensity means the c-Myc antibody molecule is easier to reach and bind with its antigen through a more spacious pathway. We thus examined the position dependence of antibody accessibility by inserting c-Myc antigen at different sites in the loop, which was used to provide the detailed structural conformation of the extracellular segment of hβ2.

In addition to the site Lys137 in the loop, c-Myc epitope was also introduced either at the C terminus or at the sites 72, 76, 114, 126, 158, and 185 in the loop. However, Lys137 is the only site with the clear c-Myc signals in either hβ2 alone or hβ2 with mslo together (Table 1). It is abnormal that we cannot see the c-Myc signals at positions 76 and 158 with coexpression of mslo with hβ2, in the presence or absence of Triton X-100 (Table 1). This may suggest that the mslo {alpha} subunit can affect the c-Myc accessibility of hβ2.


View this table:
[in this window]
[in a new window]

  		TABLE 1
Summary on locations in the P-loop of hβ2 accessible to the c-Myc epitope

 
To remove the interference of mslo, the construct {Delta}33A was selected for this purpose. In this construct, the c-Myc epitope was introduced at the positions 76, 100, 114, 126, 137, 158, and 185 in the loop, respectively. In this work, we found that the positions 126 and 137 are two easiest sites for access of antibody based on their fluorescence intensities, whereas β2 expressed alone (Fig. 5 and Table 1). It indicates that the fluorescence signal at position 126 disappears in the coexpression case. Thus, the c-Myc signal of the hβ2 loop can be covered by the {alpha} subunit, specially, by the P-loop of S5–S6 while coexpressed with {alpha} subunits.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 4.
Position dependence of the N-terminal retention signal of hβ2. A, the images for HEK293 cells transfected with hβ2–137myc or mslo+hβ2–137myc. B, the images for HEK293 cells expressed with C33 or mslo+C33 (The first 33 amino acids of hβ2 N terminus were translocated to its C terminus after Arg235.) C, the percentage intensity of hβ2, mslo+hβ2, C33, and mslo+C33 are 12.8 ± 0.9 (n = 6), 100.0 ± 0.0 (n = 95), 13.0 ± 5.6 (n = 8), and 72.4 ± 13.9 (n = 63), respectively. The scale bar indicates 5 µm.

 
Because the higher fractional intensity means the higher accessibility of the loop, the most accessible site was plotted at the highest level of the extracellular loop, the second was plotted at the second highest level, and so on. In this way, the "structural" profile of the extracellualr loop can be depicted in a letter "W," of which the apex was located around the middle of the loop (Fig. 5C). On the basis of its hydrophobicity plot (9), the hypothetical topology of hβ2 has two transmembrane domains with a large extracellular loop, of which positions 76 and 185 are just located at the N and C termini of the extracellular loop, respectively. Therefore, they should be the easier sites to be approached by antibody. Moreover, the central part of the loop is usually considered to be difficult to approach because it lays over the channel pore and deep into the cell surface (30, 31). According to the above rule of accessibility, the structural profile of the extracellualr loop can be depicted in a letter "M," of which the valley is around the middle too. To our great surprise, the accessibility plotted in Fig. 5C completely contradicts our expectation.


Figure 5
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 5.
Accessibility of the extracellular loop of hβ2 subunits. A, the images for HEK293 cells transfected with a c-Myc epitope tagged to a construct {Delta}33A at positions 76, 100, 114, 126, 158, and 185 in the extracellular loop are shown at the left panel. The scale bar indicates 5 µm. B, intensities normalized to that of {Delta}33A-137myc were plotted for mutations {Delta}33A with c-Myc epitope tags at different positions 76, 100, 114, 126, 137, 158, and 185 in the loop. The percentage intensities of {Delta}33A-76myc, {Delta}33A-100myc, {Delta}33A-114myc, {Delta}33A-126myc, {Delta}33A-137myc, {Delta}33A-158myc, and {Delta}33A-185myc are 8.8 ± 4.0 (n = 5), 44.5 ± 6.2 (n = 26), 14.5 ± 7.0 (n = 11), 97.2 ± 9.3 (n = 56), 100.0 ± 0.0 (n = 73), 36.6 ± 3.2 (n = 22), and 6.6 ± 4.8 (n = 7), respectively. C, the accessibilities of the extracellular loop by antibodies were plotted based on their fractional intensities indicated in B. Light gray circles represent the inserted c-Myc epitopes.

 


Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 6.
All of the BK channels encoded with mslo and mutations of hβ2 subunits make functional currents. All of the currents encoded by mslo and hβ2 mutation subunits were recorded in inside-out patches in presence of intracellular 10 µM Ca2+. The traces of mslo+hβ2-mutations were evoked by a voltage step from a holding potential of -180 mV to voltages ranged from -150 to 150 mV in an increment 20 mV. The scale bars represent 30 ms (time) and 2 nA (current), respectively. The voltage protocol is plotted on the upper left.

 
All Mutations of hβ2 Maintain Physiological Functions—Another way for validating the surface expression of mutations is to test currents of all the mslo+mutations by voltage clamp experiments. To make it simpler, the representative traces are only coming from some of the mutations (Fig. 6). Typically, mutations with the initial three hydrophobic residues FIW at their N termini always show a rapid inactivation with a time constant of about 25 ms at 100 mV in 10 µM Ca2+. The rest exhibits the larger inward currents at negative voltages, which indicates that there is a huge negative shift (usually more than 50 mV) in conductance-voltage (G-V) curves relative to the G-V curve of mslo {alpha} channels. The above results indicate that the hβ2 including its mutations that we constructed in this study can traffic onto the membrane surface of cells after being functionally associated with mslo {alpha} subunits.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among β-subunit trafficking, β1 subunit can traffic to the cell surface alone (14, 21), whereas β2 subunit can reach the plasma membrane with the {alpha} subunit together. Through the biotinylation experiments, Jin et al. (32) reported that the β4 subunit could target to the cell membrane based on the existence of double glycosylation sites in β4 subunits. There is no information about trafficking about β3 subunit so far. Furthermore, Zarei et al. (14, 20) observed a putative endocytic signal in the C terminus of the hβ2, which reduced the surface expression of the coexpressed hslo subunits. With polyclonal antibody against β2 subunit using the permeabilized labeling protocol, they found that the hβ2 alone appeared to reach the plasma membrane. We infer that the hβ2 alone stays on the ER membrane just underneath the plasma membrane.

In this study, we were attempting to determine a retention motif in hβ2 like the RXR in potassium inward rectifying channel (Kir) and its auxiliary SUR1 (22, 23) or the CVLF in BK splice variant (24). There are many reasons against KIR being a retention signal. For instance, the surface expression of {Delta}29A is more than that of {Delta}26A; QKIRD-hβ1 or Q23PD27P can traffic to membrane surface. Therefore, the second structure of the hβ2 N-terminus is our exclusive choice.

Using NMR spectroscope, Bentrop et al. (25) investigated the solution structure of the hβ2 N terminus (amino acids 1–45, BKβ2N). The BKβ2N structure comprises two domains connected by a flexible linker: the ball domain (formed by residues 1–17) and the chain domain (between residues 20–45) linking it to the membrane segment of hβ2. The chain domain consists of a four-turn helix including a 310-helix (between residues 18–31) with an unfolded linker at its C terminus. The BKβ2N with a four-turn helix is a properly folded peptide as it can functionally inactivate BK channels when applied to the cytoplasmic side (25). Furthermore, our results suggest that the retention signal may be derived from protein-protein or protein-lipid mechanism. Because there is no report about the specific anchoring protein of hβ2, we would rather consider a protein-lipid mechanism. From the three-dimensional structure of the four-turn helix shown in Fig. 1C (25), we found that the positively charged residues (Lys18, Arg19, and Arg26) were clustering on one side, and the hydrophobic residues (Ile21/Tyr22 and Leu30/Leu31) are lining on each side. Considering that the head group of phospholipid is negatively charged and the tail domain is hydrophobic, we infer that the resultant force coming from electric field and hydrophobic interaction may distort the helix itself to lead the hβ2 subunit coming off the membrane of the ER completely. Furthermore, the positively charged residues Lys and Arg seem to play a crucial role, because the reduction in the number of positively charged residues would somehow weaken the protein-lipid interaction.

It is well known that trypsin can attack and cleave any exposed basic residue in the hβ2 N terminus. Zhang et al. (18) reported that the first four basic residues, i.e. Arg8, Arg14, Lys18, and Arg19, had the highest trypsin accessibility of basic residue. During hβ2 assembling with mslo in the ER, the C terminus of mslo {alpha} subunit may somehow wrap the N-terminal helix of hβ2 to avoid trypsin attack (18). Correspondingly, β subunits reciprocally modulate many properties of BK {alpha} channels by the loop-loop interaction of {alpha} and β subunits. For instance, the BK {alpha}+hβ4 channel is becoming very resistant to CTX or IbTX because of the glycosylation in the loop domain of hβ4 subunits, which prevents the access of toxins to the channel vestibule (32). The outward rectification of BK {alpha}+hβ2 channels can be demolished completely by the c-Myc insert hβ2–137myc (data not shown). This study may provide us a new way to understand the interaction of the BK {alpha} subunit and β subunit.

The extracellular loop of hβ2 plays an important role in preventing the toxin from approaching the pore of BK channels (9) and producing the rectification of BK channels (19). However, the lack of the structural information about the extracellular loop prevents us from precisely determining the interacting or binding sites between the loop of hβ2 and the pore of BK channels. Our results reveal that three locations at 137, 100, and 185 represent the peak and valleys of accessibility of the extracellular loop, respectively, which may provide us with important clues to explore the interacting or binding sites in the future. In addition, four conserved cysteines influence the rectifying and pharmacological characteristics of BK channels via forming disulfide bridges to constitute a specific structure in the extracellular loop, that can be disrupted by 20 mM extracellular dithiothreitol (19). Two cysteine mutations (Cys2 and Cys3) were constructed for this purpose, whereas we did not find any changes in accessibility. Hopefully, a complete removal of four conserved cysteines may provide more clues for re-evaluating the accessibility of c-Myc antibody to the extracellular loop in the future.


   FOOTNOTES
 
* This work was supported by National Science Foundation of China Grants 30470449, 30470646, 30630020, and 30670502. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence may be addressed. Tel.: 86-10-648-884-69; Fax: 86-10-648-675-66; E-mail: xutao{at}ibp.ac.cn. 3 To whom correspondence may be addressed. Tel.: 86-27-877-921-53; Fax: 86-27-877-920-24; E-mail: jpding{at}mail.hust.edu.cn.

4 The abbreviations used are: BK, large conductance voltage- and Ca2+-activated K+; ER, endoplasmic reticulum; EGFP, enhanced green fluorescence protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Z. Q. Xu for valuable comments on the manuscript and Dr. C. L. Lingle for kindly providing us with the β1–β4 clones.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ding, J. P., Li, Z. W., and Lingle, C. J. (1998) Biophys. J. 74, 268-289[Medline] [Order article via Infotrieve]
  2. Brenner, R., Jegla, T. J., Wickenden, A., Liu, Y., and Aldrich, R. W. (2000) J. Biol. Chem. 275, 6453-6461[Abstract/Free Full Text]
  3. Brenner, R., Perez, G. J., Bonev, A. D., Eckman, D. M., Kosek, J. C., Wiler, S. W., Patterson, A. J., Nelson, M. T., and Aldrich, R. W. (2000) Nature 407, 870-876[CrossRef][Medline] [Order article via Infotrieve]
  4. Dworetzky, S. I., Boissard, C. G., Lum-Ragan, J. T., McKay, M. C., Post-Munson, D. J., Trojnacki, J. T., Chang, C. P., and Gribkoff, V. K. (1996) J. Neurosci. 16, 4543-4550[Abstract/Free Full Text]
  5. Meera, P., Wallner, M., and Toro, L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5562-5567[Abstract/Free Full Text]
  6. Nimigean, C. M., and Magleby, K. L. (1999) J. Gen. Physiol. 113, 425-440[Abstract/Free Full Text]
  7. Tseng-Crank, J., Godinot, N., Johansen, T. E., Ahring, P. K., Strobaek, D., Mertz, R., Foster, C. D., Olesen, S. P., and Reinhart, P. H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9200-9205[Abstract/Free Full Text]
  8. Wallner, M., Meera, P., and Toro, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4137-4142[Abstract/Free Full Text]
  9. Xia, X. M., Ding, J. P., and Lingle, C. J. (1999) J. Neurosci. 19, 5255-5264[Abstract/Free Full Text]
  10. Wallner, M., Meera, P., and Toro, L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14922-14927[Abstract/Free Full Text]
  11. Xia, X. M., Ding, J. P., Zeng, X. H., Duan, K. L., and Lingle, C. J. (2000) J. Neurosci. 20, 4890-4903[Abstract/Free Full Text]
  12. Li, Z. W., Ding, J. P., Kalyanaraman, V., and Lingle, C. J. (1999) J. Neurophysiol. 81, 611-624[Abstract/Free Full Text]
  13. Li, W., Gao, S. B., Lv, C. X., Wu, Y., Guo, Z. H., Ding, J. P., and Xu, T. (2007) J. Cell. Physiol. 212, 348-357[CrossRef][Medline] [Order article via Infotrieve]
  14. Toro, B., Cox, N., Wilson, R. J., Garrido-Sanabria, E., Stefani, E., Toro, L., and Zarei, M. M. (2006) Neuroscience 142, 661-669[CrossRef][Medline] [Order article via Infotrieve]
  15. Xia, X. M., Ding, J. P., and Lingle, C. J. (2003) J. Gen. Physiol. 121, 125-148[Abstract/Free Full Text]
  16. Hu, S., Labuda, M. Z., Pandolfo, M., Goss, G. G., McDermid, H. E., and Ali, D. W. (2003) Physiol. Genomics 15, 191-198[Abstract/Free Full Text]
  17. Savalli, N., Kondratiev, A., de Quintana, S. B., Toro, L., and Olcese, R. (2007) J. Gen. Physiol. 130, 117-131[Abstract/Free Full Text]
  18. Zhang, Z., Zhou, Y., Ding, J. P., Xia, X. M., and Lingle, C. J. (2006) J. Neurosci. 26, 11833-11843[Abstract/Free Full Text]
  19. Zeng, X. H., Xia, X. M., and Lingle, C. J. (2003) Nat. Struct. Biol. 10, 448-454[CrossRef][Medline] [Order article via Infotrieve]
  20. Zarei, M. M., Song, M., Wilson, R. J., Cox, N., Colom, L. V., Knaus, H. G., Stefani, E., and Toro, L. (2007) Neuroscience 147, 80-89[CrossRef][Medline] [Order article via Infotrieve]
  21. Zarei, M. M., Eghbali, M., Alioua, A., Song, M., Knaus, H. G., Stefani, E., and Toro, L. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 10072-10077[Abstract/Free Full Text]
  22. Deutsch, C. (2003) Neuron 40, 265-276[CrossRef][Medline] [Order article via Infotrieve]
  23. Zerangue, N., Schwappach, B., Jan, Y. N., and Jan, L. Y. (1999) Neuron 22, 537-548[CrossRef][Medline] [Order article via Infotrieve]
  24. Zarei, M. M., Zhu, N., Alioua, A., Eghbali, M., Stefani, E., and Toro, L. (2001) J. Biol. Chem. 276, 16232-16239[Abstract/Free Full Text]
  25. Bentrop, D., Beyermann, M., Wissmann, R., and Fakler, B. (2001) J. Biol. Chem. 276, 42116-42121[Abstract/Free Full Text]
  26. Behrens, R., Nolting, A., Reimann, F., Schwarz, M., Waldschutz, R., and Pongs, O. (2000) FEBS Lett. 474, 99-106[CrossRef][Medline] [Order article via Infotrieve]
  27. Bright, J. N., Shrivastava, I. H., Cordes, F. S., and Sansom, M. S. (2002) Biopolymers 64, 303-313[CrossRef][Medline] [Order article via Infotrieve]
  28. Kuo, A., Gulbis, J. M., Antcliff, J. F., Rahman, T., Lowe, E. D., Zimmer, J., Cuthbertson, J., Ashcroft, F. M., Ezaki, T., and Doyle, D. A. (2003) Science 300, 1922-1926[Abstract/Free Full Text]
  29. Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Nature 417, 515-522[CrossRef][Medline] [Order article via Infotrieve]
  30. Hanner, M., Vianna-Jorge, R., Kamassah, A., Schmalhofer, W. A., Knaus, H. G., Kaczorowski, G. J., and Garcia, M. L. (1998) J. Biol. Chem. 273, 16289-16296[Abstract/Free Full Text]
  31. Qian, X., Nimigean, C. M., Niu, X., Moss, B. L., and Magleby, K. L. (2002) J. Gen. Physiol. 120, 829-843[Abstract/Free Full Text]
  32. Jin, P., Weiger, T. M., and Levitan, I. B. (2002) J. Biol. Chem. 277, 43724-43729[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/5/2709    most recent
M704440200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lv, C.
Right arrow Articles by Ding, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lv, C.
Right arrow Articles by Ding, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement