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

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 a 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 similar to40% 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 initiation (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 similar to8.5-fold reduction of the surface charge density without altering any other parameter. These results indicate that the, pore plays a key role in conduction and suggest that the pore structure is not affected aspartate residue in the BKCa 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.