Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells

doi:10.21203/rs.3.rs-2480271/v1

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Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells

https://doi.org/10.21203/rs.3.rs-2480271/v1

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Voltage-gated sodium channels (VGSC) are widely expressed in various types of tumor and cancer cells, and Na_V1.5 are overexpression in highly metastatic breast cancer cells. There may be positive relations between the expression levels of Na_V1.5 and the breast cancer recurrence and metastasis. Herein, Na_V1.5 were detected and localized on the surfaces of normal and cancer breast cells by Single Molecular Recognition Imaging mode (SMRI) of Atomic Force Microscopy (AFM). The results reveal that Na_V1.5 were irregularly distributed on the surfaces of normal and cancer breast cells. The Na_V1.5 take an area percentage of 0.6% and 7.2% on normal and cancer breast cells, respectively, which indicates that there are more Na_V1.5 on cancer cells than normal cells. The specific interaction forces and binding kinetics between Na_V1.5-antibody complex system were investigated with Single Molecular Force Spectroscopy (SMFS) mode of AFM, indicating that the stability of the Na_V1.5-antibody on normal breast cells is higher than cancer breast cells. All these results will be useful to study the interactions of other ion channel-antibody systems, and will also be useful to understand the role of sodium channels in tumor metastasis and invasion.

Voltage-gated sodium channels

breast cells

Atomic Force Microscopy

Single Molecular Recognition Imaging

Single Molecular Force microscopy

VGSC are transmembrane proteins that can adjust the movements of sodium ions across cellular membranes along an electrochemical gradient [1]. Mammalian VGSC are formed by a large pseudo tetrameric pore forming α subunit (220 ~ 260 kDa) and one or two β subunits (33–36 kDa). There are nine different α subunits (Na_V1.1 ~ Na_V1.9) that show tissue specific expression profiles [1, 2]. The opening and closing of the sodium channels are regulated by the activation and inactivation gating processes, respectively. More importantly, different α subunits are widely expressed in various types of tumors and cancers, and participate in the regulation of phagocytosis, metastatic activity, cell motility and other cellular activities [3]. The dysfunction of VGSC on physiology leads to a variety of diseases and disorders [4].

In breast cancer cells, especially in highly metastatic breast cancer cells, such as MDA-MB-231 cell line, Na_V1.5 are overexpressed [5]. There are positive relations between the expression level of Na_V1.5 and the breast cancer motility, metastasis and invasiveness [6]. This indicates that Na_V1.5 may be the effective therapy targets of anticancer drugs, such as ranolazine and phenytoin [7]. Thus it has great significance and necessity in the investigations of the distributions and interactions of Na_V1.5 in normal and cancer breast cells. The mRNA and tissue sodium levels (inferred from protein-level studies, electrolyte content, clinical imaging and animal tests) have provided the evidences for the VGSC expression level in breast carcinomas and potentiation of metastasis [8]. These have also been studied by many other approaches [7]. But these investigations are from indirect means, or the spatial resolutions are limited, thus the VGSC have not been detected and localized at nanometer or single molecular spatial resolution level, the interaction forces and related binding kinetics of VGSC have not been studied at high force resolution level so far. Therefore new approaches have to be introduced in this field, the multiple and powerful nature of AFM make it the desirable toolkit in this area.

AFM, an important member of Scanning Probe Microscopy, is a powerful and versatile toolkit in biotechnology [9]. AFM can image the biological samples, such as biomacromolecules (the proteins and chromatins), cell membranes, and cells at high resolution with little damages [10–12]. SMFS mode of AFM can measure the intra- and inter molecular forces down to picoNewton level directly, meanwhile the reaction kinetics between biomolecules can also be studied. SMFS has been applied to study the specific interaction forces in antibody-antigen and receptor-ligand successfully [13, 14]. SMRI, the combination of AFM imaging and SMFS, has been developed to distinguish and locate the target biomolecules, such as the membrane proteins, antigens and receptors, in the heterogenic samples [15]. The recognition processes are confirmed to be highly specific, efficient and reproducible [16, 17].

As discussed above, the multifunction and powerful features of AFM make it an appropriate toolkit in the investigations of the distributions and interactions of Na_V1.5 in breast cells. In this work, the Na_V1.5 has been specifically localized on the cellular membranes of normal and cancer breast cells at the single molecular spatial resolution level, and the interactions (including the interaction forces and binding kinetics) between the Na_V1.5 and the anti-Na_V1.5 antibody have been studied at the picoNewton force resolution level. These studies will supply innovation methods and approaches in the investigations of ion channels on cellular membranes, and will also be useful and helpful in the further studies of other membrane proteins.

Cell culture

Hs578Bst and MDA-MB-231 cells were chosen as the representative normal and cancer breast cells, and purchased from Procell Life Science and Technology Co., Ltd (Wuhan, China). All these cells were cultured in Dulbecco’s minimum essential medium (DMEM, Biological Industries, Shanghai, China) with 10% fetal bovine serum (Biological Industries, Shanghai, China), 100 μg/mL streptomycin (Solarbio life sciences, Beijing, China) and 100 U/mL penicillin (Solarbio life sciences, Beijing, China). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37^oC in incubator, and were grown as monolayer for use.

Fluorescence staining and fluorescence microscopy

When the cells were cultured well in the petri dish, they were washed three times by phosphate buffer saline (PBS, Corning). Then the cells were fixed with 4% paraformaldehyde for 20 min, and washed with PBS for three times. The nonspecific binding sites on cells were blocked by 2% bovine serum albumin (Solarbio life sciences, Beijing, China) for 2 h. The cells were reacted with anti-Na_v1.5 polyclonal antibody (Abcam, Shanghai, China) at 4^oC overnight. Then reacted with donkey anti-goat IgG (Alexa Fluor 488, Abcam, Shanghai, China) for 1 h in darkness. Before imaging, the cells were washed with PBS for three times to remove the unreacted dye.

The fluorescence images were obtained with the laser scanning confocal microscopy LSM880 (Carl Zeiss, Shanghai, China). The Alexa Fluor 488 was excited by the laser with the wavelength of 488 nm, and the emission fluorescence (wavelength of 515 nm) was collected by the 20× or 63× objective. All the data were processed by software Zen 2.3 (Carl Zeiss, Shanghai, China).

Functionalization of the AFM tips with anti-Na_V1.5 antibody

The functionalization procedures were similar as described previously [16]. Briefly, anti-Na_V1.5 antibodies were reacted with N-succinimidyl 3-(acetylthio) propionate (SATP, Sigma-Aldrich, Shanghai, China). The cantilevers were cleaned in the O₃ atmosphere in ultraviolet radiation cleaner for 20 min to get rid of the organic contamination. Then the cantilevers were vapor treated with aminopropyltriethoxysilane (APTES, 99%, Sigma-Aldrich, Shanghai, China), and reacted with polyethylene glycol (PEG) crosslinkers (9.8 nm in length, MaL-PEG2000-NHS, JenKem Technology Co., Ltd., Beijing, China) in triethylamine (Sigma-Aldrich, Shanghai, China) and CHCl₃ (Richjoint Chemical, Shanghai, China). Then the cantilevers were immersed in 100 μg/mL anti-Na_V1.5 antibody with NaCNBH₃ (Sigma-Aldrich, Shanghai, China) as catalyst. In the last, 1 M ethanolamine (Sigma-Aldrich, Shanghai, China) was added to passivate the unreacted aldehyde groups. Then the modified tips were rinsed with PBS for two times and stored in PBS at 4°C until use.

Atomic force microscopy

All the experiments were performed with the JPK NanoWizard 4XP BioScience AFM (Bruker Corporation, Santa Barbara, California, USA). Single molecular recognition imaging was performed by the QI advanced mode, and was carried out with anti-Na_V1.5 antibody modified tips in buffer solutions at room temperature. The probes were MLCT-Bio-C (Bruker Corporation, Santa Barbara, California, USA), and the main parameters were (nominal): resonance frequency 7 kHz, spring constant 0.01 N/m. The scanning rate is 1 Hz. The recognition signals were revealed at the 75% cut-off of the background. Blocking experiments were performed by the addition of the 100 μg/mL anti-Na_V1.5 antibody into the AFM sample cell.

Force spectroscopy was operated in the contact force spectroscopy mode. The deflection sensitivity of the photo-detector was determined by the slope of the force curves captured on the surfaces of cleaning silicon wafer. The actual spring constants of the cantilevers were measured by the thermal noise method in air as described previously [18]. Thousands of force curves were obtained on various positions of different cells. Blocking experiments were performed by the addition of 100 μg/mL anti-Na_V1.5 antibody into the AFM sample cell.

All the AFM images and force curves were processed by the software JPK SPM Data Processing 7.0 (Bruker Corporation, Santa Barbara, California, USA).

The surface morphology of normal and cancer breast cells

Hs578Bst and MDA-MB-231 cells are chosen as the represent normal and cancer breast cells, respectively. In order to confirm that there are Na_V1.5 on the surfaces of cells, the Na_V1.5 on the cellular membranes of Hs578Bst and MDA-MB-231 were labeled, and the fluorescent images captured on the two types of cells are shown in Fig. 1A and 1B, respectively. Both the Hs578Bst and MDA-MB-231 cells grow well, and there are Na_V1.5 on both types of cells. All these demonstrate that the intact and healthy Hs578Bst and MDA-MB-231 cells are obtained, and there are Na_V1.5 on the surfaces of the two types of cells. Thus the two types of cells are suitable for the next further investigations.

Single molecular recognition imaging of Na_V1.5 on the surface of normal breast cells

As the resolution of fluorescence microscopy is limited, we prefer single molecular recognition imaging to investigate the distributions of Na_V1.5 at single molecular resolution. In this mode, the tips were functionalized with anti-Na_V1.5 antibody via the flexible and heterobifunctional PEG crosslinker as depicted in Fig. 2A. One anti-Na_V1.5 antibody connects only one PEG crosslinker. The nonlinear stretching characteristics of the PEG can make it to distinguish the specific events from the nonspecific ones. As the PEG is inert in chemical and physical properties, it makes the anti-Na_V1.5 antibody functionalized on the tips to reorientate rapidly and freely when the tip is approaching the surface. Meanwhile the PEG tethered anti-Na_V1.5 antibody modified on the tip can avoid to be crashed [9]. The amplitude of the cantilever is set to be less than the stretched length of the crosslinker [15]. When the Na_V1.5 sites on the cell membranes were scanned by this tip, the crosslinker will be stretched in the retraction processes of the cantilever. There will be resulting energy loss that can reduce the top peak of the oscillations. Therefore the recognition signals can be achieved and detected [19]. The recognition processes have been proved to be highly efficient, specific and reproducible [15].

The topography and corresponding recognition images captured on Hs578Bst cells are shown in Fig. 2B and 2C, respectively. The dark spots in Fig. 2C represent the recognition signals that was irregularly distributed on cell membranes. The recognition signals takes a area percentage of (0.6 ± 0.2)% (N = 5) of the cell membranes. Thus for the first time we provide the quantitative information of the amount of Na_V1.5 on cell membranes by direct means. The recognition signals were superimposed onto the topographic image (shown as the blue areas in Fig. 2B) to indicate the distributions of Na_V1.5 on the cell membranes more clearly. In order to testify that the recognition processes are specific, the blocking experiments were performed by the addition of free anti-Na_V1.5 antibody into the AFM sample cell. As the binding sites were occupied, the recognition signals disappeared as depicted in Fig. 2D. A magnified recognition site was shown in Fig. 2E. The amplitude reduction of the cantilever is about 3.4 nm according to the cross section analysis as shown in Fig. 2F. However, this value in the corresponding blocked area (Fig. 2G) is about 0.3 nm (as depicted in Fig. 2H), which indicates that the recognition signals have been blocked efficiently. There are no recognition signals on the images acquired by the bare tips or PEG modified tips (Fig. S1 in the Supplementary Information). All these demonstrate that the recognition process is highly specific and efficient.

Single molecular recognition imaging of the Na_V1.5 on the surface of cancer breast cells

In order to reveal the distribution of Na_V1.5 on cancer breast cells, SMRI was performed by anti-Na_V1.5 antibody modified tips on the surface of MDA-MB-231 cells. Figure 3A and 3C are the topography and corresponding recognition images, respectively. Figure 3B is the topography with the recognition signals superimposed. There are more recognition sites than that on Hs578Bst cells (as depicted in Fig. 2C). In order to verify the specificity of the recognition process, the blocking experiment has been performed by the addition of free anti-Na_V1.5 antibody. The recognition signals disappeared as depicted in Fig. 3D. A magnified recognition site before and after blocking is shown in Fig. 3E and 3G, respectively. The amplitude reduction has reduced from 2.6 nm (as depicted in Fig. 3F) to 0.3 nm (as indicated in Fig. 3H). The percentage of recognition areas is (7.2 ± 1.7)% (N = 5), which is about 12 times of Hs578Bst cells. This result indicates that the amount of Na_V1.5 on MDA-MB-231 cells is more than Hs578Bst cells.

Force spectroscopy between Na _V 1.5 on cell surfaces and the anti-Na _V 1.5 antibody conjugated on the AFM tips

In order to investigate the interactions between the Na_V1.5 and the anti-Na_V1.5 antibody directly, the anti-Na_V1.5 antibody was attached on the AFM tips as shown in Fig. 2A. When the tip approaches and withdraws from the surface of cells, the interaction forces between the antibody modified on the tip and the Na_V1.5 on the cell membranes can be detected and recorded as force curves. Thousands of force curves were recorded at various positions on different cells. The typical force curve acquired on the surface of Hs578Bst cells with one unbinding event is shown in Fig. 4A. The approaching and withdrawn processes are depicted as black and red curves, respectively. The distribution of unbinding forces is shown in Fig. 4C. The unbinding forces range from 20–95 pN at a loading rate of 0.4 nN/s, with the maximum distribution at 52.8 ± 10.3 pN. The binding probability (the overall force curves divided by those with the specific unbinding event) is 19.6%. After blocking with free anti-Na_V1.5 antibody, the specific unbinding force event disappeared as shown in Fig. 4B, and the binding probability has dramatically decreased to 2.8% (Fig. 4D). There are no unbinding force events in the force curves acquired with bare tips or PEG modified tips (as depicted in Fig. 4I). All these confirmed the unbinding forces between Na_V1.5 on the surface of Hs578Bst cells and anti-Na_V1.5 antibody on the tips are specifically and efficiently detected.

The typical force curves before and after blocking recorded on the surface of MDA-MB-231 cells are shown in Fig. 4E and 4F, respectively. The unbinding forces are in the range of 14–90 pN at a loading rate of 0.40 nN/s. The maximum distribution is at 35.2 ± 12.5 pN as depicted in Fig. 4G. After blocking, the specific unbinding event disappeared (as depicted in Fig. 4F). Meanwhile the binding probability has dramatically decreased from 20.3% (Fig. 4G) to 4.3% (Fig. 4H).

The unbinding force not only depends on the interactions between the Na_V1.5 and anti-Na_V1.5 antibody, but also depends on the loading rates of the AFM tip. The relation between the unbinding force and loading rate follows Eq. (1) according to the single barrier model.

$${ F}_{u}= \frac{{k}_{B}T}{{x}_{\beta }}\text{ln}\left(\frac{r{x}_{\beta }}{{k}_{B}T{k}_{off}}\right) \left(1\right)$$

where F_u is the unbinding force; k_B is the Boltzmann constant; T is the thermodynamic temperature; x_β is the separation energy barrier from the equilibrium position; r is the loading rate of the tip; k_off is the dissociation rate constant at zero force [20].

The plots of the dependence of the unbinding forces versus loading rates are shown in Fig. 4J. The green squares and red dots display the data recorded on Hs578Bst and MDA-MB-231 cells, respectively. From the fitting curves, it can be calculated that on Hs578Bst cells, x_β = 0.20 nm, k_off = 1.59 s^− 1; on MDA-MB-231 cells, x_β = 0.25 nm, k_off = 2.50 s^− 1. The dissociation rate constant on Hs578Bst cells is less than MDA-MB-231 cells, which reveals that the Na_V1.5-antibody complex on Hs578Bst cells is more stable than MDA-MB-231 cells.

Based on the transition state theory, the relations between activation energy ΔE and dissociation rate constant k_off follow Eq. (2).

$${ k}_{off}\propto \frac{-\varDelta E}{{e}^{{k}_{B}T}} \left(2\right)$$

The difference values of dissociation activation energy Δ(ΔE) of Na_V1.5-antibody complex system on Hs578Bst and MDA-MB-231 cells follows Eq. (3) [21].

$$\varDelta \left(\varDelta E\right)= -{k}_{B}T\text{l}\text{n}\left(\frac{{k}_{off}\left(Hs578Bst\right)}{{k}_{off}\left(MDA-MB-231\right)}\right) \left(3\right)$$

It can be calculated that Δ(ΔE) of Na_V1.5-antibody on Hs578Bst and MDA-MB-231 cells is 0.45k_BT. It reveals that the dissociation activation energy on Hs578Bst cells is higher than MDA-MB-231 cells, which further indicates that the Na_V1.5-antibody complex on Hs578Bst cells is more stable and harder to dissociate than MDA-MB-231 cells.

The Na_V1.5 have been detected and localized at the single molecular level on the surfaces of normal and cancer breast cells. There are more Na_V1.5 on cancer cells than normal cells (about 12 times). Thus for the first time the quantitative information on the amounts of Na_V1.5 on normal and cancer breast cells have been given. The interaction forces and binding kinetics between Na_V1.5-antibody complex system have been studied at picoNewton force resolution level. The Na_V1.5-antibody complex is stable on normal breast cells than cancer cells. The approaches and results will provide new methods in the studies of sodium channels, and will also be helpful in the further investigations of the roles of ion channels in tumor metastasis and invasion.

Acknowledgements

Not applicable.

Author contributions

XYL and LZ performed the experiments. WDZ wrote the main manuscript text. RRF, XWD, YM, YLZ, WCL and QYL prepared all figures. YHS and GWZ analyzed the data. HJZ revised the manuscript. All authors reviewed the manuscript, and read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (22004020, 61674164 and 62164001), the Natural Science Foundation of Jiangxi Province (20202ZDB01018), the open project of Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases Ministry of Education of China (XN201903), the startup funds of high-level talents of Gannan Medical University (QD202011 and QD201906) and the school level project of Gannan Medical University (YB201942).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and supplementary information files.

Consent for publication

All authors agreed to submit this manuscript.

Competing interests

The authors have declared that no competing interest exists.

Ethical Approval

Not applicable.

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No competing interests reported.

SupplementaryInformation.doc
TableofContentsTOC.png
Table of Contents (TOC) The cellular membranes of normal and cancer breast cells are scanned by the anti-Na_V1.5 antibody modified AFM tip, the Na_V1.5 can be localized at single molecular spatial resolution level, and the interactions between the Na_V1.5 and antibody can be detected at picoNewton force resolution level.

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Version 1

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You are reading this latest preprint version

Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

The surface morphology of normal and cancer breast cells

Single molecular recognition imaging of Na_V1.5 on the surface of normal breast cells

Single molecular recognition imaging of the Na_V1.5 on the surface of cancer breast cells

Force spectroscopy between Na _V 1.5 on cell surfaces and the anti-Na _V 1.5 antibody conjugated on the AFM tips

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Single Molecular Localizations of Voltage-Gated Sodium Channel NaV1.5 on the Surfaces of Normal and Cancer Breast Cells

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results And Discussion

The surface morphology of normal and cancer breast cells

Single molecular recognition imaging of NaV1.5 on the surface of normal breast cells

Single molecular recognition imaging of the NaV1.5 on the surface of cancer breast cells

Force spectroscopy between Na V 1.5 on cell surfaces and the anti-Na V 1.5 antibody conjugated on the AFM tips

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Single molecular recognition imaging of Na_V1.5 on the surface of normal breast cells

Single molecular recognition imaging of the Na_V1.5 on the surface of cancer breast cells

Force spectroscopy between Na _V 1.5 on cell surfaces and the anti-Na _V 1.5 antibody conjugated on the AFM tips