Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense.

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V(m)) increases exponentially from 21 pS at 50 mV up to 53 pS at V(m) = 200 mV, 20 degrees C. The concentration dependence of G(V(m)) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K(0.5), where G(V(m)) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work DeltaG(theta) (el) = rhoZ(g)FV(m) approximately -1.3 RT, which corresponds to the movement of one charge of the gating charge number Z(g) = 1 across the fraction rho = DeltaV(m)/V(m) = 0.15 of the membrane voltage V(m) = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (>/=5 mM, pH 6.8) with K(0.5) approximately 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.