Localization of the K(+) Lock-in and the Ba(2+) Binding Sites in a Voltage-Gated Calcium-Modulated Channel: Implications for Survival of K(+) Permeability

Using Ba(2+) as a probe, we performed a detailed characterization of an external K(+) binding site located in the pore of a large conductance Ca(2+)-activated K(+) (BK(Ca)) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba(2+) blocks BK(Ca) channels and decreasing external K(+) using a K(+) chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba(2+)-blocked events. Average Ba(2+) dwell time changes from 10 s at 10 mM external K(+) to 100 ms in the limit of very low [K(+)]. Using a model where external K(+) binds to a site hindering the exit of Ba(2+) toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549–568), we calculated a dissociation constant of 2.7 μM for K(+) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K(+)] is reduced below 4 μM using the crown ether. Channel activity can be recovered by adding K(+), Rb(+), Cs(+), or NH(4) (+) to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K(+)] is due to K(+) binding to a site having a very high affinity. Occupancy of this site by K(+) avoids the channel conductance collapse and the exit of Ba(2+) toward the external side. External tetraethylammonium also reduced the Ba(2+) off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K(+). It is explained in terms of a model in which the conduction pore contains Ba(2+), K(+), and tetraethylammonium simultaneously, with the K(+) binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69–77) imply that the lock-in site and the Ba(2+) sites are the external and internal ion sites of the selectivity filter, respectively.