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Neutralization of Aspartate 292 Reduces Long Channel Openings and Gating Current Slow Component
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
Address correspondence to Riccardo Olcese, Department 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
Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca2+-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173–184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by 
20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a 40% reduction of outward single channel conductance, as well as a strong outward rectification.
Key Words: potassium channel • conduction • gating current • pore • hSlo
Abbreviations used in this paper: HP, holding potential; SHP, subtracting holding potential.
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  R. A. Piskorowski and R. W. Aldrich Relationship between Pore Occupancy and Gating in BK Potassium Channels J. Gen. Physiol., April 24, 2006; 127(5): 557 - 576. [Abstract] [Full Text] [PDF]
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T. Haug, D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese Regulation of K+ Flow by a Ring of Negative Charges in the Outer Pore of BKCa Channels. Part I: Aspartate 292 modulates K+ Conduction by External Surface Charge Effect J. Gen. Physiol., July 26, 2004; 124(2): 173 - 184. [Abstract] [Full Text] [PDF]
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