Skip to main content
SpringerLink
Log in

Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells

  • Sensory Physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Cholesterol is an essential component of cell membranes, and determines their rigidity and fluidity. Alterations in membrane cholesterol by MβCD or water-soluble cholesterol affect the stiffness, capacitance, motility, and cell length of outer hair cells (OHCs). This suggests that reconstruction of the cytoskeleton may be induced by cholesterol alterations. In this study, we investigated intracellular signaling pathways involving G proteins to determine whether they modulate the changes in voltage-dependent capacitance caused by cholesterol alterations. Membrane capacitance of isolated guinea pig OHCs were assessed using a two-sine voltage stimulus protocol superimposed onto a voltage ramp (200 ms duration) from −150 to +140 mV. One group of OHCs was treated with 100 μM guanosine 5′-O-(3-thiotriphosphate) tetralithium salt (GTPγS), the GTP analog, administrated into individual cells via patch pipettes. Another group of OHCs was internally perfused with 600 μM guanosine 5′-(β-thio) diphosphate trilithium salt (GDPβS), the GDP analog. A third group was perfused with internal solution only as a control. Application of 1 mM MβCD shifted non-linear capacitance curves to the depolarized direction of the control group with reduction of the peak capacitance (C mpeak). After the 10-min application of MβCD, shifts of voltage at C mpeak (V cmpeak) and reduction of C mpeak were 73.32 ± 11.09 mV and 9.09 ± 2.10 pF, respectively (n = 4). On the other hand, in the GTPγS-treated group, the shift of V cmpeak and reduction of C mpeak were attenuated remarkably. The shift of V cmpeak and reduction of C mpeak in the 10-min application of MβCD were 9.73 ± 10.92 mV and 3.08 ± 1.91 pF, respectively (n = 7). MβCD decreased the cell length by 16.53 ± 4.27 % in the control group and by 6.45 ± 6.22 % in the GTPγS group. In addition, we investigated the effects of GDPβS on cholesterol-treated OHCs. One millimolar cholesterol was externally applied after the 4-min application of 1 mM MβCD because the shift of V-C m function caused by cholesterol alone was small. Application of cholesterol shifted V-C m curves of the control group to the hyperpolarized direction with increase of the C mpeak. After the 10-min application of cholesterol, changes of V cmpeak and C mpeak were −9.19 ± 6.68 mV and 2.14 ± 0.44 pF, respectively (n = 4). On the other hand, in the GDPβS-treated OHCs, the shift of V cmpeak and increase of C mpeak were attenuated markedly. The shift of V cmpeak and increase of C mpeak after 10 min were 5.13 ± 10.46 mV and −0.55 ± 1.39 pF, respectively (n = 6). This study demonstrated that internally perfused GTPγS inhibited the MβCD effects and GDPβS inhibited the cholesterol effects, raising the possibility that G proteins may be involved in outer hair cell homeostasis as well as the possibility that cholesterol response may be G protein mediated. More study is required to clarify the detailed role of G proteins in the relation between cholesterol and the OHC cytoskeleton.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

OHC:

Outer hair cell

MβCD:

Methyl-beta-cyclodextrin

NLC:

Nonlinear capacitance

C m :

Membrane capacitance

C mpeak :

Peak capacitance

V m :

Membrane potential

V cmpeak :

Voltage at peak capacitance

Q max :

Maximal charge movement

z :

Valence

C lin :

Linear membrane capacitance

GTPγS:

Guanosine 5′-O-(3-thiotriphosphate) tetralithium salt

GDPβS:

Guanosine 5′-(β-thio) diphosphate trilithium salt

References

  1. Abe T, Kakehata S, Kitani R, Maruya S, Navaratnam D, Santos-Sacchi J, Shinkawa H (2007) Developmental expression of the outer hair cell motor prestin in the mouse. J Membr Biol 215:49–56

    Article  PubMed  CAS  Google Scholar 

  2. Ashmore J, Avan P, Brownell WE, Dallos P, Dierkes K, Fettiplace R, Grosh K, Hackney CM, Hudspeth AJ, Julicher F, Lindner B, Martin P, Meaud J, Petit C, Sacchi JR, Canlon B (2010) The remarkable cochlear amplifier. Hear Res 266:1–17

    Article  PubMed  CAS  Google Scholar 

  3. Ashmore J (2008) Cochlear outer hair cell motility. Physiol Rev 88:173–210

    Article  PubMed  CAS  Google Scholar 

  4. Belyantseva IA, Adler HJ, Curi R, Frolenkov GI, Kachar B (2000) Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells. J Neurosci 20:116

    Google Scholar 

  5. Beseničar M, Bavdek A, Kladnik A, Maček P, Anderluh G (2007) Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin: a surface plasmon resonance approach. Biochim Biophys Acta 1778:175–184

    PubMed  Google Scholar 

  6. Bodmer D, Brors D, Pak K, Gloddek B, Ryan A (2002) Rescue of auditory hair cells from aminoglycoside toxicity by Clostridium difficile toxin B, an inhibitor of the small GTPases Rho/Rac/Cdc42. Hear Res 172:81–86

    Article  PubMed  CAS  Google Scholar 

  7. Brownell WE, Spector AA, Raphael RM, Popel AS (2001) Micro- and nanomechanics of the cochlear outer hair cell. Annu Rev Biomed Eng 3:169–194

    Article  PubMed  CAS  Google Scholar 

  8. Brownell WE, Jacob S, Hakizimana P, Ulfendahl M, Fridberger A (2011) Membrane cholesterol modulates cochlear electromechanics. Pflugers Arch - Eur J Physiol 461:677–686

    Article  CAS  Google Scholar 

  9. Dallos P (2008) Cochlear amplification, outer hair cells and prestin. Curr Opin Neurobiol 18:370–376

    Article  PubMed  CAS  Google Scholar 

  10. Dallos P, Wu X, Cheatham MA, Gao J, Zheng J, Anderson CT, Jia S, Wang X, Cheng WH, Sengupta S, He DZ, Zuo J (2008) Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 58:333–339

    Article  PubMed  CAS  Google Scholar 

  11. He DZZ, Zheng J, Kalinec F, Kakehata S, Santos-Sacchi J (2006) Tuning in to the amazing outer hair cell: membrane wizardry with a twist and shout. J Membr Biol 209:119–134

    Article  PubMed  CAS  Google Scholar 

  12. Homma K, Dallos P (2010) Evidence that prestin has at least two voltage-dependent steps. J Biol Chem 286:2297–2307

    Article  PubMed  Google Scholar 

  13. Jiang H, Sha SH, Schacht J (2006) Rac/Rho pathway regulates actin depolymerization induced by aminoglycoside antibiotics. J Neurosci Res 83(8):1544–1551

    Article  PubMed  CAS  Google Scholar 

  14. Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92:145–159

    Article  PubMed  CAS  Google Scholar 

  15. Huang G, Santos-Sacchi J (1994) Motility voltage sensor of the outer hair cell resides within the lateral plasma membrane. Proc Natl Acad Sci U S A 91:12268–12272

    Article  PubMed  CAS  Google Scholar 

  16. Kachar B, Brownell WE, Altschuler R, Fex J (1986) Electrokinetic shape changes of cochlear outer hair cells. Nature 322:365–8

    Article  PubMed  CAS  Google Scholar 

  17. Kakehata S, Santos-Sacci J (1995) Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge. Biophys J 68:2190–2197

    Article  PubMed  CAS  Google Scholar 

  18. Kakehata S, Santos-Sacchi J (1996) Effects of salicylate and lanthanides on outer hair cell motility and associated gating charge. J Neurosci 16:4881–4889

    PubMed  CAS  Google Scholar 

  19. Kalinec F, Holley MC, Iwasa KH, Lim DJ, Kachar B (1992) A membrane-based force generation mechanism in auditory sensory cells. Proc Natl Acad Sci U S A 89:8671–8675

    Article  PubMed  CAS  Google Scholar 

  20. Kalinec F, Zhang M, Urrutia R, Kalinec G (2000) Rho GTPases mediate the regulation of cochlear outer hair cell motility by acetylcholine. Biochemistry 275:28000–28005

    CAS  Google Scholar 

  21. Katanaev V, Wymann M (1998) GTPgamma S-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor. J Cell Sci 111:1583–1594

    PubMed  CAS  Google Scholar 

  22. Khatibzadech K, Gupta S, Farrell B, Brownell WE, Anvari B (2012) Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane. Soft Matter 8:8350–8360

    Article  Google Scholar 

  23. Kitani R, Kakehata S, Kalinec F (2011) Motile responses of cochlear outer hair cells stimulated with an alternating electrical field. Hear Res 280:209–218

    Article  PubMed  Google Scholar 

  24. Kitani R, Kakehata S, Murakoshi M, Wada H, Maruya S, Abe T, Shinkawa H (2008) The direct effects of cholesterol and Mβcd on outer hair cell motility and capacitance. ARO abstract828

  25. Matsumoto N, Kalinec F (2005) Prestin-dependent and prestin-independent motility of guinea pig outer hair cells. Hear Res 208:1–13

    Article  PubMed  CAS  Google Scholar 

  26. Matsumoto N, Kitani R, Maricle A, Mueller M, Kalinec F (2010) Pivotal role of actin depolymerization in the regulation of cochlear outer hair cell motility. Biophys J 99:2067–2076

    Article  PubMed  CAS  Google Scholar 

  27. Matsumoto N, Kitani R, Kalinec F (2011) Linking LIMK1 deficiency to hyperacusis and progressive hearing loss in individuals with Williams syndrome. Commun Integr Biol 4:208–210

    Article  PubMed  CAS  Google Scholar 

  28. Meri F (2005) Lipid rafts and regulation of the cytoskeleton during T cell activation. 360:1663–1672

  29. Nguyen TV, Brownell WE (1998) Contribution of membrane cholesterol to outer hair cell lateral wall stiffness. Otolaryngol Head Neck Surg 119:14–20

    Article  PubMed  CAS  Google Scholar 

  30. Pivola U, Xing-Qun L, Virkkala J, Saarma M, Murakata C, Camoratto M, Walton K, Ylikoski J (2000) Rescue of hearing, auditory hair cells, and neurons by CEP-1347/KT7515, an inhibitor of c-Jun N-terminal kinase activation. 20(1):43–50

  31. Preyer S, Baisch A, Bless D, Gummer AW (2001) Distortion product otoacoustic emissions in human hypercholesterolemia. Hear Res 152:139–151

    Article  PubMed  CAS  Google Scholar 

  32. Rajagopalan L, Greeson JN, Xia A, Liu H, Sturm A, Raphael RM, Davidson AL, Oghalai JS, Pereira FA, Brownell WE (2007) Tuning of the outer hair cell motor by membrane cholesterol. J Biol Chem 282:36659–36670

    Article  PubMed  CAS  Google Scholar 

  33. Rajagopalan L, Organ-Darling LE, Liu H, Davidson AL, Raphael RM, Brownell WE, Pereira FA (2010) Glycosylation regulates prestin cellular activity. J Assoc Res Otolaryngol 11:39–51

    Article  PubMed  Google Scholar 

  34. Rong J, Shapiro M, Trogan E, Fisher E (2003) Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad Sci U S A 100:13531–13536

    Article  PubMed  CAS  Google Scholar 

  35. Santos-Sacci J (1991) Reversible inhibition of voltage-dependent outer hair cell motility and capacitance. J Neurosci Off J Soc Neurosci 100:13531–13536

    Google Scholar 

  36. Santos-Sacci J (2003) New tunes from Corti’s organ: the outer hair cell boogie rules. Curr Opin Neurobiol 13:459–468

    Article  Google Scholar 

  37. Sfondouris J, Rajagopalan L, Pereira FA, Brownell WE (2008) Membrane composition modulates prestin-associated charge movement. J Biol Chem 283:22473–22481

    Article  PubMed  CAS  Google Scholar 

  38. Sturm AK, Rajagopalan L, Yoo D, Brownell WE, Pereira FA (2007) Functional expression and microdomain localization of prestin in cultured cells. Otolaryngol Head Neck Surg 136:434–439

    Article  PubMed  Google Scholar 

  39. Tolomeo J, Steele C, Holley M (1996) Mechanical properties of the lateral cortex of mammalian auditory outer hair cells. Biophys J 71:421–429

    Article  PubMed  CAS  Google Scholar 

  40. Wang A, Yang S, Jia S, He DZ (2010) Prestin forms oligomer with mechanically independent subunits. Brain Res 33:28–35

    Article  Google Scholar 

  41. Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P (2000) Prestin is the motor protein of cochlear outer hair cells. Nature 405:149–155

    Article  PubMed  CAS  Google Scholar 

  42. Zigmond S, Joyce M, Borleis J, Bokoch G, Devreotes P (1997) Regulation of actin polymerization in cell-free systems by GTPgS and Cdc42. J Cell Biol 138:363–374

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (S.K). The authors would like to thank Ms. A. C. Apple-Mathews for valuable suggestions concerning English usage.

Author information

Authors and Affiliations

  1. Department of Otorhinolaryngology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan

    Takahiko Nagaki, Seiji Kakehata, Rei Kitani, Takahisa Abe & Hideichi Shinkawa

  2. Department of Otolaryngology Head and Neck Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida Nishi, Yamagata, 990-9585, Japan

    Seiji Kakehata

Authors
  1. Takahiko Nagaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seiji Kakehata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rei Kitani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takahisa Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hideichi Shinkawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seiji Kakehata.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nagaki, T., Kakehata, S., Kitani, R. et al. Effects of cholesterol alterations are mediated via G-protein-related pathways in outer hair cells. Pflugers Arch - Eur J Physiol 465, 1041–1049 (2013). https://doi.org/10.1007/s00424-013-1230-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-013-1230-3

Keywords

Search

Navigation