|
|
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voltage-dependent Charge Movement and Gating Currents
2 Department of Physiology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095
3 Centro de Estudios Científicos, Valdivia, Chile
Address correspondence to Ana M. Correa, Department of Anesthesiology, David Geffen School of Medicine at University of California, Los Angeles, BH-509A, CHS Box 957115, Los Angeles, CA 90095-7115. Fax: (310) 794-9612; email: nani{at}ucla.edu
The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure–function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372–2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at –90 mV while holding at –150 mV. Charge–voltage (Q–V) curves showed sigmoidal dependence on voltage with gating charge saturating at –10 mV. Charge movement was shifted by –22 mV relative to the conductance–voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 µs observed for a change in preconditioning voltage from –160 to –80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q–V curves were shifted by approximately –60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.
Key Words: sodium channel • gating charge • bacterial channels • C-type inactivation • Cole-Moore shift
Abbreviations used in this paper: HP, holding potential; MS, methane sulfonate; NMG, N-methyl-D-glucamine.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. M. Gamal El-Din, D. Grogler, C. Lehmann, H. Heldstab, and N. G. Greeff More Gating Charges are Needed to Open a Shaker K+ Channel than are Needed to Open an rBIIA Na+ Channel Biophys. J., August 1, 2008; 95(3): 1165 - 1175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lewis, V. Jogini, L. Blachowicz, M. Laine, and B. Roux Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K+ Channel of Known Structure J. Gen. Physiol., May 26, 2008; 131(6): 549 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fujinami, T. Sato, J. S. Trimmer, B. W. Spiller, D. E. Clapham, T. A. Krulwich, I. Kawagishi, and M. Ito The voltage-gated Na+ channel NaVBP co-localizes with methyl-accepting chemotaxis protein at cell poles of alkaliphilic Bacillus pseudofirmus OF4 Microbiology, December 1, 2007; 153(12): 4027 - 4038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ouyang, T.-Y. Jih, T.-T. Zhang, A. M. Correa, and H. C. Hemmings Jr. Isoflurane Inhibits NaChBac, a Prokaryotic Voltage-Gated Sodium Channel J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 1076 - 1083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Pavlov, C. Bladen, R. Winkfein, C. Diao, P. Dhaliwal, and R. J. French The Pore, not Cytoplasmic Domains, Underlies Inactivation in a Prokaryotic Sodium Channel Biophys. J., July 1, 2005; 89(1): 232 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
