Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System

Abstract

Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BK_Ca channel. The properties of BK_Ca channel, such as voltage-dependence, Ca²⁺ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BK_Ca channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca²⁺ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BK_Ca channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BK_Ca channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

INTRODUCTION

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel plays significant physiological roles in various cell types of different tissues, such as neurons, smooth muscles, secretary glands and cartilages.¹⁾ It has been well known that BK_Ca channel consists of pore-forming α subunit (BKα) and auxiliary β subunit (BKβ). BKα is encoded by a single gene, whereas four subtypes of BKβ (BKβ1–β4) have been reported to give tissue-specific modifications to BK channel activities by their tissue-dependent expression.²⁾ Recently leucine-rich-repeat-containing (LRRC) proteins have been identified as novel additional auxiliary BKγ subunits (BKγ).³⁾ LRRC26, LRRC52, LRRC55 and LRRC38 have been specifically re-named as the BKγ1, BKγ2, BKγ3 and BKγ4 for BK_Ca channels, respectively.⁴⁾ The incorporation of BKγ to BK_Ca channel complex promotes a voltage-shift of channel activation to very negative membrane potentials, even without an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).^3,5) In addition, BKγ1 can also modulate pharmacological features of BK_Ca channels.⁶⁾ At present, BKγ1 has been known to function as a BK_Ca channel auxiliary subunit in parotid acinar cells,^6,7) airway epithelial cells^8,9) and bronchial and vascular smooth muscle cells (SMCs).^5,10)

It has been also reported that BKγ regulates BK_Ca channel functions in an all-or-none manner.¹¹⁾ This implies that only one BKγ interacts with BKα tetramer can fully activate the BK_Ca channel activity. In contrast, two independent studies recently suggested that a BK_Ca channel can incorporate up to four BKγ1 using fluorescence resonance energy transfer (FRET) analysis and single channel recordings¹²⁾ or lanthanide resonance energy transfer.¹³⁾ However, these studies do not directly count the number of BKγ1 within a single functional BK_Ca channel. In addition, they used Xenopus oocytes that have different protein expression system from mammalian cells.¹⁴⁾ In the present study we directly counted BKγ1 within a single functional BK_Ca channel expressed in a human-derived cell line, HEK293 cells, using single molecular imaging with a total internal reflection fluorescence (TIRF) microscope.

MATERIALS AND METHODS

Cell Culture

The HEK293 cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan). Cells were suspended in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20 U/mL penicillin, and 20 µg/mL streptomycin (Sigma-Aldrich, U.S.A.) at 37°C.

TIRF Imaging

Single molecule imaging was performed using a TIRF imaging system with an objective lens (CFI Apo TIRF 100 × /1.49, oil immersion; Nikon, Tokyo, Japan) as described previously.^15,16) Data were collected with an EM-CCD camera and analyzed by AQUACOSMOS software (Hamamatsu Photonics, Hamamatsu, Japan). Green fluorescent protein (GFP)- or mCherry-fused protein targets were excited with a 488-nm argon laser (Spectra-Physics, Santa Clara, CA, U.S.A.) or a 543-nm He/Ne laser (Melles Griot, Carlsbad, CA, U.S.A.), respectively. GFP/mCherry emission data were collected using a combination of dichroic mirrors and dual band-pass filters (505–530/570–660 nm; Omega Optical, Brattleboro, VT, U.S.A.). The resolution of images was 105 nm per pixel (x–y) and 200 nm (z). All experiments were carried out at room temperature (25°C).

Plasmid Constructs and Transfection

Full-length of cDNA encoding the human KCNMA1 (BKα, NM_002247) was subcloned into mCherry-N1 (BKα-mCherry) (Clontech Laboratories, Mountain View, CA, U.S.A.). Human KCNMB1 (BKβ1, NM_004137) or LRRC26 (BKγ1, NM_001013653) were also subcloned into pAcGFP-N1 (BKβ1-GFP and BKγ1-GFP). The fusion construct in which N-terminus of BKγ1-GFP is fused to the C-terminus of BKα-mCherry (BKα + BKγ1) was also established to realize equal expression of BKα and BKγ1 within a HEK293 cell.³⁾ In this construct, BKγ1-GFP is cleaved at N-terminal signal peptide sequences, separated from BKα-mCherry at ER and exported to plasma membrane.³⁾ All constructs were confirmed by DNA sequencing. HEK293 cells were transiently transfected with cDNA (each 1 µg for co-expression) using LipofectAMINE 2000 (Invitrogen, Waltham, CA, U.S.A.). Experiments were preformed 24 to 48 h after transfection. It has been confirmed that the functions or properties of these constructs tagged with fluorescent proteins are equivalent to those of native constructs.^3,5,15)

Electrophysiological Recordings

Electrophysiological studies were performed using conventional whole-cell voltage-clamp and current-clamp techniques with a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan), an analog-digital converter (DIGIDATA 1440A; Molecular Devices, Sunnyvale, CA, U.S.A.) and a pCLAMP software (version 10.2; Molecular Devices), as described previously.¹⁶⁾ For measurements of whole-cell BK_Ca channel currents, the pipette filling solution contained (mM); 140 KCl, 0.265 CaCl₂, 4 MgCl₂, 10 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 2 Na₂ATP and 5 ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (pCa8.0). The pH was adjusted to 7.2 with 1 M KOH. The extracellular solution had an ionic composition of (mM) 137 NaCl, 5.9 KCl, 2.2 CaCl₂, 1.2 MgCl₂, 14 glucose and 10 HEPES. The pH was adjusted to 7.4 with NaOH. BK_Ca channel currents were activated from a holding potential of −80 mV by applying 80-ms voltage steps to voltages between −100 and +100 mV in increments of 10 mV. All experiments were carried out at room temperature (25°C). The total cell membrane capacitance was measured to confirm single cell recording and for calculation of current density per capacitance of unit membrane area (BKα, 27.2 ± 1.0 pF, n = 4; BKα + γ1, 20.4 ± 1.9 pF, n = 4).

Single Molecule GFP Bleaching

The number of BKγ1 subunits in the immediate vicinity of the plasma membrane of HEK293 cells was counted by observing the bleaching steps of GFP fused to the subunits in a single particle, as described previously.^16,17) Images were obtained in the order of mCherry and GFP to avoid bleaching by cross-excitation. Images were acquired at 10 Hz for 100–120 s. Fluorescence intensity in a region of interest (ROI) (5 × 5 pixels) was calculated by subtracting the background in 24 pixels around the ROI. The number of bleaching steps was determined by eye from the fluorescence signal trace. The distributions of observed number of bleaching steps were compared with zero-truncated binomial distribution, P (x, n, p) = _nC_x p^x (1−p)^n−x, where n is the number of subunits (n = 4 for tetramers), x is the observed number of bleaching steps, and p is the apparent probability for GFP fluorescence.¹⁸⁾

Reagents

Paxilline (Alomone Laboratories) was dissolved in dimethyl sulfoxide (DMSO) as a 1 mM stock solution. The final concentration of DMSO was 0.1%. Stock solutions were stored −20°C prior to use.

Statistics

Pooled data are presented as the mean ± standard error of the mean (S.E.M.) Statistical significance between two groups was evaluated using t-test after F-test application. When the distribution between the two groups was equal, Student’s t-test was used. If not, Welch’s t-test was utilized for statistics analysis. p < 0.05 was considered statistically significant.

RESULTS

BK_Ca Channel Currents of HEK 293 Cells Expressing BKα and BKγ1

When BKα labeled with mCherry or BKα + BKγ1 labeled with GFP were transiently expressed in HEK293 cells, outward currents were recorded upon depolarization under voltage clamp (Fig. 1A). BKα labeled with mCherry shows a typical BK channel single channel conductance (approximately 230 pS) and it was equivalent to that of non-labeled BKα.¹⁵⁾ [Ca²⁺]_i in the pipette solution was kept low at pCa 8.0 (i.e., 10 nM). The depolarization-induced currents were inhibited by a BK_Ca channel blocker, 1 µM paxilline. In the BKα expressing cells, 1 µM paxilline-sensitive outward currents were detected only at much higher voltages than that in BKα + BKγ1 (Figs. 1A, B). As shown in Fig. 1C, the paxilline-sensitive current density in BKα + BKγ1 at +50 mV was markedly larger than that in BKα (BKα: 1.3 ± 2.2 pA/pF; n = 4, BKα + BKγ1: 93.4 ± 23.4 pA/pF; n = 4, p = 0.0294 vs. BKα). These results showed that BKα and BKγ1 labeled with mCherry and GFP possess function to elicit paxilline-sensitive and voltage-dependent outward currents and that co-expression of BKγ1 with BKα extensively enhanced the current amplitude under the experimental conditions ([Ca²⁺]_i = 10 nM). Importantly, BKγ1 labeled with GFP enhanced BK_Ca channel activity to the same extent as non-labeled BKγ1 does.^3,5)

Fig. 1. BK_Ca Channel Currents of HEK 293 Cells Expressing BKα and BKγ1

(A) Typical traces of outward currents from HEK293 cells expressing BKα or BKα + BKγ1 before (control) and after the treatment with 1 µM paxilline, a selective BK_Ca channel blocker. BKα and BKγ1 were labeled with mCherry and GFP, respectively. Holding potential was −80 mV. Voltage steps from −100 to +100 mV in 10 mV increments were given to cells. The Ca²⁺ concentration in the pipette filling solution was fixed at pCa8.0. (B) The relationship between the density of paxilline-sensitive current and activation voltage in BKα (n = 4) and BKα + BKγ1 (n = 4) is shown. The current density was obtained by dividing the amplitude of peak outward current with cell membrane capacitance. (C) The paxilline-sensitive current density at +50 mV in BKα (n = 4) and BKα + BKγ1 (n = 4) is compared.

BKγ1 Stably Forms Molecular Complexes with BKα in the Plasma Membrane

The stability of BKα-BKγ1 complex and also the effect of molecular complex formation on BKα mobility in the plasma membrane were measured by visualizing BKα-mCherry and BKγ1-GFP in live cells using single molecular imaging system with a TIRF microscope (Fig. 2A). When BKα + BKγ1 was expressed in HEK293 cells, these proteins can be visualized as dot-like fluorescent signals. BKα-mCherry alone showed dynamic mobility on the membrane surface in HEK293 cells (Fig. 2B). As shown in Figs. 2B and C, some BKα-mCherry particles got colocalized with BKγ1-GFP on the membrane surface. During the measurements for 60 s, two particles kept moving together on the plasma membrane. Furthermore, the mobility of BKα-mCherry was significantly decreased by the colocalization with BKγ1-GFP (distribution efficiency (D_60s, µm²/s): BKα-mCherry alone 0.052 ± 0.013, n = 20; BKα-mCherry colocalized with BKγ1-GFP 0.010 ± 0.006, n = 10, p = 0.0406 vs. BKα-mCherry alone, Fig. 2D). These data directly demonstrate that BKγ1 form a stable complex with BKα and BKγ1 markedly stabilizes BKα movement in the plasma membrane of living cells.

Fig. 2. The Mobility of BKα Was Decreased by Interaction with BKγ1

The localization and behavior of BKα and BKγ1 in plasma membrane were visualized by using a TIRF microscope. BKα alone or BKα + BKγ1 (BKα-mCherry and BKγ1-GFP) was transiently expressed in HEK293 cells. (A) Typical TIRF image of HEK293 cells expressing BKα + BKγ1. (B) The particles of BKα-mCherry and BKγ1-GFP on the membrane surface, indicated by the image in A (particle a and b), were tracked for 60 s. (C) Molecular behavior of BKα colocalized with BKγ1 particle was imaged every 5 s. Note that BKα and BKγ1 moved around together in a very limited area during the recording for over 60 s. (D) Diffusion coefficient (D_60s) values of BKα interacting with BKγ1 (BKα + BKγ1) (n = 10) or BKα alone (n = 20) are shown.²³⁾ (Color figure can be accessed in the online version.)

BKγ1 Form BK_Ca Channel Complex with BKα in a 1 : 1 Stoichiometry in Native Mammalian Cells

The stoichiometry between BKα and BKγ1 within a functional BK_Ca channel complex was studied using a single molecule GFP bleaching method^16,17) (Fig. 3A). In this method, bleaching steps of GFP signal was counted in a visualized auxiliary subunit (BKγ1 or BKβ1), that was colocalized with mCherry-labeled BKα detected as a merged fluorescent signal. It has been shown that one to four BKβ1 can bind to a BKα tetramer and increase the channel activity depending on its stoichiometry.¹⁹⁾ Therefore, we used BKβ1 as a control in this experiment.

Fig. 3. Four BKγ1 Can Be Incorporated in a Single BK_Ca Channel Complex

BKα + BKγ1 or BKα-mCherry and BKβ1-GFP (transfection ratio was 1 : 1) were transiently expressed in HEK293 cells. Based on GFP bleaching method, the bleaching steps of GFP signals in a TIRF region were counted for determining the number of BKβ1 or BKγ1 within a single fluorescent particle. (A) Typical TIRF images of HEK293 cells expressing BKα + BKγ1 are shown. Analyzed particles are indicated by white circles. Only GFP particles colocalized with mCherry particles were analyzed. (B, C) Changes in GFP fluorescence intensity of BKβ1-GFP (B) and BKγ1-GFP (C) are presented. Solid lines indicate bleaching step(s). Dotted line indicates complete bleaching (basal) level. (D) The number of bleaching steps of BKβ1-GFP and BKγ1-GFP were summarized. The blue bars indicate the binominal distribution expected for tetramers with probability of 0.75 for a single GFP to be fluorescent. (Color figure can be accessed in the online version.)

First, HEK293 cells co-expressing BKα-mCherry and BKβ1-GFP were prepared by transfection of cDNA at a ratio of 1 : 1. The colocalization ratio of BKα-mCherry and BKβ1-GFP was 86% (BKα alone: 14.2 ± 4.5% and BKα colocalized with BKβ1: 85.8 ± 4.5%, 119 particles from 12 cells). Single spots of BKβ1-GFP colocalized with BKα-mCherry displayed mainly 3 or 4 step bleaching (1 step, 14.5%; 2 steps, 20.3%; 3 steps, 37.5%; 4 steps, 25%, 53 particles from 12 cells, Figs. 3B, D). The population distribution of the data obtained from this bleaching step analysis was well fitted by the theoretical binomial distribution for a tetramer (n = 4), with the apparent probability of GFP being fluorescent during excitation (p) set at 0.75¹⁸⁾ (see “Materials and Methods”). This result provided evidence suggesting that each GFP spot contained four BKβ1 under these experimental conditions.

Second, HEK293 cells co-expressing BKα-mCherry and BKγ1-GFP were prepared by the transfection with BKα + BKγ1 plasmid. The colocalization ratio of BKα-mCherry and BKγ1-GFP was 81% (BKα alone: 19.1 ± 4.5% and BKα colocalized with γ1: 80.9 ± 4.5%, 194 particles from 7 cells). Similarly, the BKγ1-GFP showed a distribution that peaked at 3 or 4 steps (1 step, 10.5%; 2 steps, 15.8%; 3 steps, 47.4%; 4 steps, 26.3%, 43 particles from 7 cells) (Figs. 3C, D). This distribution was also well fitted by binomial distribution, given tetramer (n = 4) and apparent fluorescence probability of p = 0.75. Taken together, BKγ1 can form tetramers with BKα at the stoichiometry of 1 : 1 in a similar manner as BKβ1.

DISCUSSION

So far, two groups have suggested that a tetrameric BKα complex can interact with up to four BKγ1 using imaging and functional analyses.¹²⁾ However, they did not directly count the number of BKγ1 within a single functional BK_Ca channel. Furthermore, their data largely rely on results obtained from oocytes whose biochemical machinery does not always assemble mammalian proteins faithfully. In the present study, we directly counted BKγ1 within a single functional BK_Ca channel expressed in a human-derived cell line, HEK293 cells, using a TIRF microscope. Our data demonstrate that BKγ1 can stably interact with BKα at the stoichiometry of 1 : 1 within a single BK_Ca channel complex in HEK293 cells.

We previously reported that the mobility of BKα was markedly reduced by forming complexes with BKβ1.¹⁷⁾ Accordingly, we examined here whether BKγ1 also influences the mobility of BKα on the membrane surface. As shown in Fig. 2, BKα mobility was significantly reduced by forming complex with BKγ1 (Fig. 2). These results suggest that BKγ1 molecule interacts with scaffold proteins via intracellular C-terminal, while the molecular entity of the scaffold proteins was not further examined in this study. As for BKβ1, it dynamically changes its intracellular localization in response to intracellular signaling of Ca^{2+ 20)} or NO²¹⁾ in SMCs and regulates BK_Ca channel activity. The movement of BKγ1 between intracellular compartment and plasma membrane with respect to its influence on BK_Ca channel activity is an emergent topic and remains to be examined.

As shown in Fig. 3D, the distribution pattern of numbers of BKβ1 and BKγ1 was well fitted by binomial distribution under the condition where “n” and apparent fluorescence probability p are 4 and 0.75, respectively. Given that fluorescence probability of GFP and colocalization ratio (86 and 81% in BKα + BKβ1 and BKα + BKγ1 expressing cells, respectively) are equal in these two types of cells and the expression efficacy was comparable and not changed by co-expression, the binding affinity between BKγ1 and BKα is supposed to be comparable with that between BKβ1 and BKα. It has been suggested that a BK_Ca channel can contain zero to four BKβ or BKγ per channel. The incorporation of one to four BKβ shows incremental effects on BK_Ca channel gating.¹⁹⁾ In contrast, it has been suggested that BKγ1 affects BK_Ca channel gating in an all-or-none manner and that a single BKγ1 incorporation is enough to induce the full channel activation. However, the researchers used BKβ2-γ1 chimeric construct but not the native γ1 subunit.¹¹⁾ The present study, in which equal amount of cDNA of pore and auxiliary subunits are transfected in the expression system, showed that four BKβ1 or BKγ1 can be the most preferentially incorporated in each BK_Ca channel complex.

BKβ1 is thought to be a major BK_Ca channel subunit in smooth muscle tissues, such as vascular²²⁾ and bronchial²³⁾ smooth muscles. Recently we demonstrated that BKγ1 expression is exceptionally high in mouse bronchial SMCs in comparison with other several types of SMCs and that the half activation voltage (V_1/2) of BK_Ca channel current is close to that in BKα + BKγ1 in HEK293 reconstituted system.⁵⁾ The physiological impact of the finding is that BK_Ca channel activity extensively contributes to keep the stable resting membrane potential at well negative to −60 mV in bronchial SMCs. Moreover, we found that the additional co-expression of BKβ1 to BKα + BKγ1 in HEK293 cells (BKα + BKβ1 + BKγ1) unexpectedly resulted in a slight sift of V_1/2 to positive direction. Considering that both BKβ1 and BKγ1 are expressed in mouse bronchial SMCs, it is possible that both subunits simultaneously bind to the same BKα tetramer. In the previous and present studies, however, it was not quantitatively examined how BKβ1 and BKγ1 can be incorporated together in a single BK_Ca channel. Thus, the hot issue about the relationship between BKβ and BKγ, i.e., how these subunits influence the binding to BKα each other, remains to be further determined.

Acknowledgments

This research was supported by JSPS KAKENHI Grant Numbers, 16H06215, 16K15127, and 19H03381 to Y.S., 16K08278, 17H05537, and 19K07125 to H.Y., 26293021, 16K15128, 15H01408, and 18KK0218 to Y.I. This work was also supported by a Grant-in-Aid from Salt Science Research Foundation [Grant 1637] to Y.S.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Cui J, Yang H, Lee US. Molecular mechanisms of BK channel activation. Cell. Mol. Life Sci., 66, 852–875 (2009).
• 2) Contreras GF, Castillo K, Enrique N, Carrasquel-Ursulaez W, Castillo JP, Milesi V, Neely A, Alvarez O, Ferreira G, Gonzalez C, Latorre R. A BK (Slo1) channel journey from molecule to physiology. Channels, 7, 442–458 (2013).
• 3) Yan J, Aldrich RW. LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium. Nature, 466, 513–516 (2010).
• 4) Yan J, Aldrich RW. BK potassium channel modulation by leucine-rich repeat-containing proteins. Proc. Natl. Acad. Sci. U.S.A., 109, 7917–7922 (2012).
• 5) Noda S, Suzuki Y, Yamamura H, Giles WR, Imaizumi Y. Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca²⁺-activated K⁺ channels in bronchial smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol., 318, L366–L375 (2020).
• 6) Almassy J, Begenisich T. The LRRC26 protein selectively alters the efficacy of BK channel activators. Mol. Pharmacol., 81, 21–30 (2012).
• 7) Yang C, Gonzalez-Perez V, Mukaibo T, Melvin JE, Xia XM, Lingle CJ. Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells. Proc. Natl. Acad. Sci. U.S.A., 114, E3739–E3747 (2017).
• 8) Manzanares D, Srinivasan M, Salathe ST, Ivonnet P, Baumlin N, Dennis JS, Conner GE, Salathe M. IFN-γ-mediated reduction of large-conductance, Ca²⁺-activated, voltage-dependent K⁺ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol., 306, L453–L462 (2014).
• 9) Manzanares D, Krick S, Baumlin N, Dennis JS, Tyrrell J, Tarran R, Salathe M. Airway surface dehydration by transforming growth factor β (TGF-β) in cystic fibrosis is due to decreased function of a voltage-dependent potassium channel and can be rescued by the drug pirfenidone. J. Biol. Chem., 290, 25710–25716 (2015).
• 10) Evanson KW, Bannister JP, Leo MD, Jaggar JH. LRRC26 is a functional BK channel auxiliary γ subunit in arterial smooth muscle cells. Circ. Res., 115, 423–431 (2014).
• 11) Gonzalez-Perez V, Xia XM, Lingle CJ. Functional regulation of BK potassium channels by γ1 auxiliary subunits. Proc. Natl. Acad. Sci. U.S.A., 111, 4868–4873 (2014).
• 12) Gonzalez-Perez V, Ben Johny M, Xia XM, Lingle CJ. Regulatory γ1 subunits defy symmetry in functional modulation of BK channels. Proc. Natl. Acad. Sci. U.S.A., 115, 9923–9928 (2018).
• 13) Carrasquel-Ursulaez W, Alvarez O, Bezanilla F, Latorre R. Determination of the stoichiometry between α- and γ1 subunits of the BK channel using LRET. Biophys. J., 114, 2493–2497 (2018).
• 14) McGuire H, Aurousseau MR, Bowie D, Blunck R. Automating single subunit counting of membrane proteins in mammalian cells. J. Biol. Chem., 287, 35912–35921 (2012).
• 15) Suzuki Y, Ohya S, Yamamura H, Giles WR, Imaizumi Y. A new splice variant of large conductance Ca²⁺-activated K⁺ (BK) channel α subunit alters human chondrocyte function. J. Biol. Chem., 291, 24247–24260 (2016).
• 16) Suzuki Y, Yamamura H, Ohya S, Imaizumi Y. Caveolin-1 facilitates the direct coupling between large conductance Ca²⁺-activated K⁺ (BK_Ca) and Ca_v1.2 Ca²⁺ channels and their clustering to regulate membrane excitability in vascular myocytes. J. Biol. Chem., 288, 36750–36761 (2013).
• 17) Yamamura H, Ikeda C, Suzuki Y, Ohya S, Imaizumi Y. Molecular assembly and dynamics of fluorescent protein-tagged single KCa1.1 channel in expression system and vascular smooth muscle cells. Am. J. Physiol. Cell Physiol., 302, C1257–C1268 (2012).
• 18) Ulbrich MH, Isacoff EY. Subunit counting in membrane-bound proteins. Nat. Methods, 4, 319–321 (2007).
• 19) Wang YW, Ding JP, Xia XM, Lingle CJ. Consequences of the stoichiometry of Slo1 α and auxiliary β subunits on functional properties of large-conductance Ca²⁺-activated K⁺ channels. J. Neurosci., 22, 1550–1561 (2002).
• 20) Leo MD, Zhai X, Muralidharan P, Kuruvilla KP, Bulley S, Boop FA, Jaggar JH. Membrane depolarization activates BK channels through ROCK-mediated beta1 subunit surface trafficking to limit vasoconstriction. Sci. Signal., 10, eaah5417 (2017).
• 21) Leo MD, Bannister JP, Narayanan D, Nair A, Grubbs JE, Gabrick KS, Boop FA, Jaggar JH. Dynamic regulation of β1 subunit trafficking controls vascular contractility. Proc. Natl. Acad. Sci. U.S.A., 111, 2361–2366 (2014).
• 22) Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, Aldrich RW. Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature, 407, 870–876 (2000).
• 23) Semenov I, Wang B, Herlihy JT, Brenner R. BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle. Am. J. Physiol. Lung Cell. Mol. Physiol., 291, L802–L810 (2006).