Localization of the K+ lock-in and the Ba2+ binding sites in a voltage-gated calcium-modulated channel - Implications for survival of K+ permeability

Using Ba2+ as a probe, we performed a detailed characterization of an exter nal K+ binding site located in the pore of a large conductance Ca2+-activat ed K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bi layers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces t he duration of the Ba2+-blocked events. Average Ba2+ dwell time changes fro m 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 Ba2+ toward the external side (Neyton, J., and C. Miller 1988. J: Gen. Physiol. 92:549 -568), we calculated a dissociation constant of 2.7 mu M for K+ at this loc k-in site. We also found that BKCa channels enter into a long-lasting nonco nductive state when the external [K+] is reduced below 4 mu M using the cro wn ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4 to the external solution. These results suggest that the BKCa channel stab ility 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 conduc tance collapse and the exit of Ba2+ toward the external side. External tetr aethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires di e presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. A ltogether; 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. Chai t, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivi ty filter, respectively.