Spermine Block of th e Strong Inward Rectifier Potassium Channel Kir2.1: Dual Roles of Surface Charge Screening and Pore Block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg 2 . Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers. key words: K channels • inward rectification • polyamine • spermine • modeling I N T R O D U C T I O N Inward rectifier potassium (Kir) channels conduct inward current efficiently at potentials negative to the K reversal potential (E K ), but progressively less so as membrane potential approaches or exceeds E K . The inward rectifying property plays an essential role in stabilizing resting membrane potential and regulating excitability in many cell types (Hille, 2001; Doupnik et al., 1995). For strong inward rectifiers in the Kir2 family, the mechanism underlying strong inward rectification involves voltage-dependent block by Mg 2 and polyamines (Matsuda et al., 1987; Vandenberg, 1987; Ficker et al., 1994; Lopatin et al., 1994; Fakler et al., 1995). Mutagenesis studies have identified negatively charged residues in two different regions which are critical for strong inward rectification: D172 in the M 2 region (Lu and MacKinnon, 1994; Stanfield et al., 1994; Wible et al., 1994; Yang et al., 1995) and E224 and E299 in the cytoplasmic COOH terminus (Taglialatela et al., 1995; Yang et al., 1995; Kubo and Murata, 2001). Block by polyamines has a complex dependence on both voltage and concentration. A quantitative model of polyamine block has been described by Lopatin et al. (1995). In their “long-pore plugging” model, they assumed two spermine molecules sequentially enter the channel pore. The model reproduced the shallow voltage-dependent component of block with rapid kinetics due to the first spermine entering the pore, and the steeply voltage-dependent component of block with slower kinetics corresponding to a second spermine molecule entering the pore and interacting with the first spermine molecule. Subsequently, they were able to simplify the model somewhat by postulating that some of the steep voltage dependence was related to polyamines sweeping K ions from the pore (Pearson and Nichols, 1998). In a previous study, we performed competition experiments using the polyamine toxin philanthotoxin, which structurally resembles polyamine at one end, but has a large hydrophobic group at the other end (Lee et al., 1999). The results, as well as those of a recent study by Kubo and Murata (2001), suggested a model in which positively charged polyamines such as spermine can bind to a region of the channel involving the negatively charged residues E224 and E299 without occluding the pore. Kubo and Murata (2001) argued that the two negative charges function as an intermediate binding site for spermine near the inner vestibule, which then facilitates entry of spermine to a binding site occluding ion permeation loAddress correspondence to James N. Weiss, Division of Cardiology, Rm. 3645, MRL Building, UCLA School of Medicine, Los Angeles, CA 90095. Fax: (310) 206-5777; E-mail: jweiss@mednet.ucla. edu