Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion.
We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.
Prod'hom
B
B
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Pietrobon
D
D
Hess
P
P
D000818:Animals; D001464:Barium / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002414:Cations, Monovalent / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004553:Electric Conductivity; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D011522:Protons; D012964:Sodium / Q000494:pharmacology
D015220:Calcium Channels; D002414:Cations, Monovalent; D011522:Protons; D001464:Barium; D008094:Lithium; D012964:Sodium