Flow by a Ring of Negative Charges in the Outer Pore of BK Ca Channels . Part I : Aspartate 292 modulates K Conduction by External Surface Charge Effect
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The pore region of the majority of K channels contains the highly conserved GYGD sequence, known as the K channel signature sequence, where the GYG is critical for K selectivity (Heginbotham, L., T. Abramson, and R. MacKinnon. 1992. Science . 258:1152–1155). Exchanging the aspartate residue with asparagine in this sequence abolishes ionic conductance of the Shaker K channel (D447N) (Hurst, R.S., L. Toro, and E. Stefani. 1996. FEBS Lett . 388:59–65). In contrast, we found that the corresponding mutation (D292N) in the pore forming subunit (hSlo) of the voltageand Ca 2 -activated K channel (BK Ca , MaxiK) did not prevent conduction but reduced single channel conductance. We have investigated the role of outer pore negative charges in ion conduction (this paper) and channel gating (Haug, T., R. Olcese, T. Ligia, and E. Stefani. 2004. J . Gen Physiol . 124: 185–197). In symmetrical 120 mM [K ], the D292N mutation reduced the outward single channel conductance by 40% and nearly abolished inward K flow (outward rectification). This rectification was partially relieved by increasing the external K concentration to 700 mM. Small inward currents were resolved by introducing an additional mutation (R207Q) that greatly increases the open probability of the channel. A four-state multi-ion pore model that incorporates the effects of surface charge was used to simulate the essential properties of channel conduction. The conduction properties of the mutant channel (D292N) could be predicted by a simple 8.5-fold reduction of the surface charge density without altering any other parameter. These results indicate that the aspartate residue in the BK Ca pore plays a key role in conduction and suggest that the pore structure is not affected by the mutation. We speculate that the negative charge strongly accumulates K in the outer vestibule close to the selectivity filter, thus increasing the rate of ion entry into the pore. key words: MaxiK channel • conduction • permeation • surface charge • Markov model I N T R O D U C T I O N Early biophysical work explained ion selectivity and conductance in K channels by a multi-ion single file pore containing more than one ion binding site (Neyton and Miller, 1988a; Neyton and Miller, 1988b; Harris et al., 1998; Hille, 2001). This initial hypothesis of a K channel pore with multiple K binding was directly visualized after resolving the crystal structure of bacterial K channels (Doyle et al., 1998; Zhou et al., 2001; Jiang et al., 2003) and has been recently supported by molecular dynamics (MD) simulation (Berneche and Roux, 2001; Berneche and Roux, 2003; Capener et al., 2003). Although the amino acid sequence of the pore forming subunit of the BK Ca channel shows high similarity with other K selective channels (Adelman et al., 1992; Butler et al., 1993; Wallner et al., 1995; Brelidze et al., 2003; Shealy et al., 2003; Nimigean et al., 2003), it conducts K 20-fold better (200–300 pS vs. 5–15 pS) while maintaining a high selectivity for K (Latorre et al., 1989). Recent work demonstrates that negatively charged residues in the inner vestibule of the mammalian BK Ca (mSlo) channel pore contribute to its high conductance. The neutralization of these negative charges in the mSlo channel (E321N/E324N) produces a twofold reduction in single channel conductance (Brelidze et al., 2003; Nimigean et al., 2003). Accordingly, K channels with smaller conductances (i.e., KcsA and Shaker ) lack negative charges at equivalent positions. Thus, one would expect that negatively charged residues in the outer vestibule could also be involved in K conAddress correspondence to Riccardo Olcese, Dept. of Anesthesiology, Division of Molecular Medicine, BH-570 CHS, David Geffen School of Medicine, Box 95711, University of California, Los Angeles, Los Angeles, CA 90095-7115. Fax: (310) 206-1947; email: rolcese@ucla.edu T. Haug’s present address is Dept. of Molecular Biosciences, University of Oslo, NO-0316 Oslo, Norway. Abbreviations used in this paper: MD, molecular dynamics; PMF, potential of mean force; WT, wild-type. on O cber 5, 2017 jgp.rress.org D ow nladed fom