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Aspartate 292 modulates K+ Conduction by External Surface Charge Effect
2 Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
3 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
4 Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
5 Department of Nuclear Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073
Address 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{at}ucla.edu
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 voltage- and Ca2+-activated K+ channel (BKCa, 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 BKCa 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
Abbreviations used in this paper: MD, molecular dynamics; PMF, potential of mean force; WT, wild-type.
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