External Barium Block of Shaker Potassium Channels - BioMedSearch

External Barium Block of Shaker Potassium Channels: Evidence for Two Binding Sites R. S. HURST,* R. LATORRE,$ L. TORO,* and E. STEFANI* From the *Department of Anesthesiology, University of California at Los Angeles, Los Angeles, California 90095; and the *Universityof Chile and Centro de Estudios Cientificos de Santiago, Santiago, Chile ABSTRACT External barium ions inhibit K + currents of Xenopus oocytes expressing ShH4 A6-46, the non-inactivating deletion of the Shaker K + channel. At the macroscopic level, Ba 2+ block comprises both a fast and a slow component. The fast c o m p o n e n t is less sensitive to Ba 2+ (apparent dissociation constant at 0 mV, /~0), ~" 19.1 mM) than the slow c o m p o n e n t and is also less voltage dependent (apparent electrical distance, 8, ~ 0.14). The slow c o m p o n e n t (/~0), ~ 9.4 mM, 8 0.25) is relieved by outward K + current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast c o m p o n e n t of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unhlocking reaction. In contrast, the slow c o m p o n e n t appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (~) produced by 2 m M B a ~+ is , ' d 5 9 s (holding potential, HP = - 9 0 mV). However, the blocking rate of this slow component is not a linear function of external Ba 2+ and tends to saturate at higher concentrations. This is inconsistent with a simple hi-molecular blocking reaction. These features of external Ba ~+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow c o m p o n e n t of block. INTRODUCTION Barium ions are only slightly larger in crystal radius than K § yet they can block the p o r e of m a n y voltage-gated K + channels (e.g., A r m s t r o n g a n d Taylor, 1980; Eaton a n d Brodwick, 1980; G r i m m e r a n d Cahalan, 1989; WoUmuth, 1994). W h e n applied to the internal side o f the squid axon-delayed rectifier, Ba 2+ binds at a site that is accesible only when the channel is o p e n , well within the m e m b r a n e field (Armstrong a n d Taylor, 1980; Eaton a n d Brodwick, 1980). Molecular biological a p p r o a c h e s using cloned voltage-gated K + channels are providing insight to the structural deterAddress correspondence to Raymond S. Hunt, Department of Anesthesiology, BH-612 CHS, University of California at Los Angeles, Los Angeles, CA 90095-1778. j. GEN.PHYSIOL.• The RockefellerUniversityPress. 0022-1295/95/12/1087/19 $2.00 Volume 106 December1995 1069-1087

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minants governing internal Ba 2+ block. For example, a single a m i n o acid substitution within the putative p o r e - f o r m i n g region of DRK1, a cloned delayed rectifier, was shown to decrease the dissociation rate of internal Ba 2+ by m o r e than 10 times (Taglialatela, Drewe, and Brown, 1993). Similarly, the sixth putative m e m b r a n e spanning region ($6) and two positions within the $4-$5 loop have b e e n d e m o n strated to contribute to internal Ba 2+ block of the Shaker K § channel (Slesinger, Jan, and Jan, 1993; Lopez, Jan, a n d Jan, 1994). Barium ions can also block voltage-gated K § channels when applied to the extracellular side of the m e m b r a n e (Armstrong and Taylor, 1980; Armstrong, Swenson, and Taylor, 1982; Grissmer a n d Cahalan, 1989; Wollmuth, 1994). Unlike internal block, external Ba 2+ block o f the squid axon-delayed rectifier does not require channel opening, even though the binding site resides within the K § conduction pathway (Armstrong a n d Taylor, 1980; A r m s t r o n g et al., 1982). In contrast, external Ba 2+ blocks the T-lymphocyte voltage-dependent K + channel only after the channel has o p e n e d (Grissmer a n d Cahalan, 1989). T h e effect of extracellular Ba z+ on the M-like voltage-dependent K § current of rod p h o t o r e c e p t o r s is m o r e complicated. In this case, Ba 2+ alters the voltage sensitivity and kinetics of the channel as well as reducing the c o n d u c t a n c e (Wollmuth, 1994). T h e present work extends the study of external Ba 2+ block to the cloned Shaker K § channel. It provides evidence that the Shaker channel has at least two external Ba ~+ blocking sites. O n e is a relatively low affinity site with an a p p a r e n t dissociation constant at 0 mV(K