Molecular basis of inward rectification: Polyamine interaction sites located by combined channel and ligand mutagenesis

Polyamines cause inward rectification of (Kir) K channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter. key words: inward rectifier • spermine • diamine • rectification • selectivity filter I N T R O D U C T I O N Sir Bernard Katz discovered inward rectification of potassium channels over 50 yr ago (Katz, 1949). The defining features of classical strong inward rectification, very steep voltage dependence and strict dependence on external [K ] (Hagiwara and Takahashi, 1974; Hagiwara and Yoshii, 1979), underlie multiple physiological phenomena, including stable electrical activity in the heart and neuronal cells, potassium siphoning in glia, and blood [K ] control of vascular tone (Newman, 1993; von Beckerath et al., 1996; Lopatin and Nichols, 2001). The realization, almost 10 yr ago, that naturally occurring polyamines (spermine, spermidine, and putrescine) are responsible for strong rectification immediately led us to propose that these agents induce rectification by “long pore plugging,” in which polyamines enter deeply into the single filing region of the channel in competition with K ions (Nichols and Lopatin, 1997). Such a mechanism could intuitively underlie the steepness of the voltage dependence of pore block, particularly if K ions were obligatorily displaced through the pore, and hence through the membrane field, in the blocking process (Ruppersberg et al., 1994; Pearson and Nichols, 1998; Guo et al., 2003). Furthermore, this mechanism could give rise to a strict dependence on external [K ] if polyamines reached sites that were directly sensitive to binding of K ions from the outside of the membrane (Park and Miller, 1992; Lopatin and Nichols, 1996). Molecular modeling of the Kir pore, using the crystal structures of MthK and KcsA as templates, has recently led us to propose that strong rectification may result from polyamine interaction within the selectivity filter itself (Dibb et al., 2003; Rose and Nichols, 2003). We propose that negative charges in the inner cavity, which control the strength of rectification (Nichols and Lopatin, 1997), act by reducing the energetic barrier to entry into the selectivity filter, and that excess charge movement (i.e., greater than the charge on the blocking polyamine) is then due to the obligatory displacement of K ions from inner cavity and selectivity filter sites to the outside of the membrane (Rose and Nichols, 2003). Guo and colleagues (Guo and Lu, 2003; Guo et al., 2003) have recently proposed a quite different location of polyamine block during strong rectification. Address correspondence to Colin G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Fax: 314-362-7463; email: [email protected] H.T. Kurata and L.R. Phillips contributed equally to this work. Abbreviations used in this paper: DA9, 1,9 diamino-nonane; DAn, (1,n)diaminoalkanes; WT, wild type. on S etem er 7, 2009 jgp.rress.org D ow nladed fom Published October 11, 2004