Time - irreversible Subconductance Gating Associated with Ba 2 1 Block of Large Conductance Ca 2 1 - activated K 1 Channels

Ba 2 1 block of large conductance Ca 2 1 -activated K 1 channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba 2 1 ion would dissociate to the external solution (150 mM N -methyld -glucamine 1 o , 500 mM K 1 i , 10 m M Ba 2 1 i , 1 30 mV, and 100 m M Ca 2 1 i to fully activate the channel), Ba 2 1 blocks with a mean duration of z 2 s occurred, on average, once every z 100 ms of channel open time. Of these Ba 2 1 blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at z 0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in z 10,000 Ba 2 1 blocks. Thus, the preopenings represent Ba 2 1 -induced time-irreversible subconductance gating. The fraction of Ba 2 1 blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean 5 0.75 ms) appeared independent of changes in [Ba 2 1 ] i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at 1 10 mV to 0.39 at 1 90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba 2 1 was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba 2 1 o and no added Ba 2 1 i . Adding K 1 , Rb 1 , or Na 1 to the external solution decreased the fraction of Ba 2 1 blocks with preopenings, with K 1 and Rb 1 being more effective than Na 1 . These results are consistent with models in which the blocking Ba 2 1 ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba 2 1 blocking site and acts directly as the preopening gate. key words: barium block • skeletal muscle • microscopic reversibility • asymmetric gating • preopening i n t r o d u c t i o n Blocking ions have served as useful probes to gain insight into the structure of the conducting pores of K 1 channels (Armstrong, 1971, 1975; Armstrong et al., 1982; Yellen, 1984 a , 1984 b ; Ferguson, 1991; Lucchesi and Moczydlowski, 1991; Slesinger et al., 1993; Taglialatela et al., 1993; Hurst et al., 1995; Sohma et al., 1996; Baukrowitz and Yellen, 1996). One blocking ion that has yielded a great deal of information about the pore of large conductance Ca 2 1 -activated K 1 (BK) 1 channels is Ba 2 1 (Vergara and Latorre, 1983; Benham et al., 1985; Miller, 1987; Miller et al., 1987; Neyton and Miller, 1988 a , 1988 b ; Neyton and Pelleschi, 1991; Diaz et al., 1996). Ba 2 1 enters and blocks the pore of the open channel in a voltage-dependent manner from either side of the membrane, with much higher concentrations of Ba 2 1 required for the same blocking rate from the external as from the internal solution. Ba 2 1 typically remains bound to the blocking site, situated 30–50% through the electric field from the inside, for several seconds. Ba 2 1 then dissociates, restoring the current to the level observed before the block. With positive membrane potentials and the impermeant ion N -methyld -glucamine 1 (NMDG 1 ) replacing external K 1 , Ba 2 1 typically dissociates to the outside solution. Adding external K 1 , which can bind to external lock-in and enhancement sites, can force dissociation of the blocking Ba 1 to the internal solution (Neyton and Miller, 1988 a , 1988 b ). From a series of experiments examining the effects of external and internal ions and membrane potential on the duration of Ba 1 blocks, Neyton and Miller (1988 b ) suggested that there are at least four K 1 binding sites within the pore of the BK channel that can be simultaneously occupied: an internal lock-in site located 30% through the electric field from the inside, an external enhancement site located 50% through the electric field, the Ba 1 blocking site located between the internal lock-in and the external enhancement sites, and an external lock-in site located 85% through the electric field from the inside. The impetus for our study was the chance observation by Ferguson et al. (1994) that Ba 2 1 blockade may be more complex than detailed above. They found that, when all the external K 1 was replaced with Address correspondence to Dr. Karl L. Magleby, Department of Physiology and Biophysics R-430, University of Miami School of Medicine, Miami, FL 33101-6430. Fax: 305-243-6898; E-mail: kmagleby@mednet. med.miami.edu 1 Abbreviations used in this paper: BK channel, Ca 2 1 -activated K 1 channel; NMDA, N -methyld -glucamine. on Jne 1, 2017 D ow nladed fom Published February 1, 1998