Voltage-Dependent Proton Transport by the Voltage Sensor of the ShakerK+ Channel

AbstractIn voltage-dependent ion channels, pore opening is initiated by electrically driven movements of charged residues, and this movement generates a gating current. To examine structural rearrangements in the Shaker K+ channel, basic residues R365 and R368 in the S4 segment were replaced with histidine, and gating currents were recorded. Changes in gating charge displacement with solvent pH reveal voltage-dependent changes in exposure of the histidine to solvent protons. This technique directly monitors accessibility changes during gating, probes the environment even in confined locations, and introduces minimal interference of gating charge motion. The results indicate that charges 365 and 368 traverse the entire electric field during gating. The remarkable implication of the successive exposure of histidine to each side of the membrane is that in a pH gradient, the voltage sensor transports protons

Gating of the Bacterial Sodium Channel, NaChBac: Voltage-dependent Charge Movement and Gating Currents

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

Lifeact-mEGFP Reveals a Dynamic Apical F-Actin Network in Tip Growing Plant Cells

Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin\u27s role in tip growing plant cells