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Original Article

Implications for Survival of K⁺ Permeability

Cecilia Vergara^a,b, Osvaldo Alvarez^a,b, and Ramon Latorre^a,b,c

^a From the Departmento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
^b Centro de Estudios Científicos de Santiago, Casilla 16443, Santiago 9, Chile
^c Department of Anesthesiology, University of California Los Angeles, Los Angeles, California 90095-1778
Centro de Estudios Científicos de Santiago, Casilla 16443, Las Condes, Santiago 9, Chile.Fax: 562-233-8336;

ramon{at}cecs.cl

Using Ba²⁺ as a probe, we performed a detailed characterization of an external K⁺ binding site located in the pore of a large conductance Ca²⁺-activated K⁺ (BK_Ca) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba²⁺ blocks BK_Ca channels and decreasing external K⁺ using a K⁺ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba²⁺-blocked events. Average Ba²⁺ 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 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 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 NH₄ ⁺ 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 Ba²⁺ toward the external side. External tetraethylammonium also reduced the Ba²⁺ 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 Ba²⁺, 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 Ba²⁺ sites are the external and internal ion sites of the selectivity filter, respectively.

Key Words: K_Ca channel • multiple occupancy • barium block • tetraethylammonium • lipid bilayer

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