Although ion permeation and gating of L-type Ca2+ channels are generally considered separate processes controlled by distinct components of the channel protein, ion selectivity can vary with the kinetic state. To test this possibility, we studied single-channel currents (cell-attached) of recombinant L-type channels (Ca(v)1.2, beta(2a), and alpha(2)delta) transiently expressed in tsA201 cells in the presence of the channel agonist BayK 8644 which promotes long channel openings (Mode 2 openings). We found that both the brief (Mode 1) and long (Mode 2) mean open times in the presence of Ca2+ were relatively longer than those with Ba2+. The unitary slope conductance with Ba2+ was significantly larger (p < 0.05) in Mode 2 openings than for brief Mode 1 openings, whereas the conductance with Ca2+ did not vary with mode gating. Consequently, the gamma(Ba):gamma(Ca) ratio was greater for Mode 2 than Mode 1 openings. Our findings indicate that both ion permeation and gating kinetics of the L-type channel are differentially modulated by permeable ions. Ca2+ binding to the L-type channel may stabilize the alteration of channel ion permeability mediated by gating kinetics, and thus, play a role in preventing excessive ion entry when the activation gating of the channel is promoted to the prolonged open state. (c) 2005 Elsevier Inc. All rights reserved.