Haug T et al. (2004), Regulation of K+ flow by a ring of negative cha...

XB-ART-3231

J Gen Physiol 2004 Aug 01;1242:173-84. doi: 10.1085/jgp.200308949.

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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.

Haug T , Sigg D , Ciani S , Toro L , Stefani E , Olcese R .

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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 alpha subunit (hSlo) of the voltage- and Ca(2+)-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 approximately 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 approximately 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.

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Species referenced: Xenopus laevis
Genes referenced: kcnma1 tbx2

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	Figure 1. . The D292N mutation neutralizes the negatively charged aspartic acid in the GYGD selectivity filter of the pore. The top panel shows the amino acid sequence alignment corresponding to the pore regions of hSlo and Shaker K+ channels. The boxes enclose identical amino acids, while gray background shows residues with conserved properties. (A) Two of the four monomers that constitute a functional K+ channel based on the crystal structure of KcsA proposed by Doyle et al. (1998). A probable arrangement of the conserved sequence of the pore region corresponding to the selectivity filter is shown. (B) The arrangement of all four monomers viewed from the top, revealing the central pore of the conduction pathway and the position of the four aspartates (red) lining the pore vestibule.
	Figure 2. . Single channel conductance reduction by the D292N mutation. Single channel currents were recorded from excised inside-out patches of Xenopus oocytes injected with hSlo (A) and hSlo-D292N (C). 40-ms voltage pulses to various potentials (numbers at left) were applied from 0-mV holding potential. The closed state indicated with arrows and the open channel current level is indicated with a dotted line. The recordings are done in symmetrical 120 mM K+ solutions with contaminant Ca2+. (B and D) Single channel IâV curves (hSlo, n = 5, and hSlo-D292N, n = 7). The outward conductance of the hSlo-D292N channel is approximately half of the conductance of the hSlo channel.
	Figure 3. . Drastic reduction of inward currents by the D292N mutation. (A) Current recordings from inside-out patches of oocytes expressing hSlo (left) and hSlo-D292N (right) channels. The voltage protocol is shown above the current traces. Note the lack of detectable single channel tail currents for the mutant channels (right). Linear components were subtracted using traces without channel openings. (B) Macroscopic current recordings from hSlo (left), hSlo-D292N (middle), and uninjected oocyte (right). The current was elicited by the voltage protocol shown above the traces, with a P/4 subtraction protocol from â90 mV subtracting holding potential. Note the smaller tail current amplitude of hSlo-D292N when compared with hSlo.
	Figure 4. . Detection of single channel openings in the hSlo-R207Q-D292N mutant. Single channel current recordings at selected potentials from inside-out patches in symmetrical 120 mM K+ and contaminant Ca2+. (A and B) hSlo-R207Q and hSlo-R207Q-D292N channel activity during 40-ms voltage pulses to various potentials from HP = 0 mV. Linear components were subtracted using traces without channel openings. The closed channel current level is indicated with arrows, and the open channel current level is indicated with a dotted line. Average single channel IâV curves for hSlo-R207Q (n = 3) and hSlo-R207Q-D292N (n = 5) are shown in B and D, respectively.
	Figure 5. . Effect of symmetric [K+] on single channel conductance in hSlo and hSlo-D292N channels. Single channel current recordings from inside-out patches of oocytes expressing hSlo (A) and hSlo-D292N (B) are shown in various symmetrical [K+] in contaminant Ca2+. The current is elicited with 40-ms steps to +50 mV (A and B) and â50 mV (D) from 0-mV holding potential. The patch in B contains at least three channels. Note in D inward channel openings in hSlo-D292N at â50 mV and 700 mM symmetrical [K+]. Linear components were subtracted using traces without channel openings. (C and E) Single channel IâV curves for hSlo and hSlo-D292N, respectively. No inward single channels were detected in hSlo-D292N in 50 (â¡) or 200 (âµ) mM K+. (F) Single channel conductance vs. [K+] plot for hSlo (â) and hSlo-D292N (â¡) channels. The single channel conductance was estimated from the linear range of the IâV relationship between +50 and +100 mV. The ionic strength of the solution was not kept constant, but increased with [K+].
	Figure 6. . A conduction model for hSlo and hSlo-D292N channels. (A) State diagram for a cyclic four-state model illustrating the metastable states of pore occupancy. The selectivity filter contains five K+ binding sites; K+ ions are depicted as green spheres. (B) Schematic free energy landscape for both hSlo and hSlo-D292N channels in standard conditions (symmetric 120 mM K+, no surface charge, and zero potential) during one permeation cycle. The landscape illustrates the free energy of the entire system âpore + ionsâ in different state of occupancy. The minima (G0â3) are to the energies of the metastable states S0â3. The maxima (G01â12â23â03) are the transition energies. Results of model fitting to experimental data are shown in C and D. (C) Experimental (symbols) and fitted (lines) single channel IâV curves for WT hSlo (red) and hSlo-D292N (blue) at indicated symmetrical [K+]. (D) Experimental (symbols) and fitted (lines) single channel chord conductance vs. [K+] for hSlo (red) and hSlo-D292N (blue). The insert shows the model parameters. Î±0 is the preexponential factor; G0â3 are the free energies of the states S0â3; G01â12â23â03 are the transition state energies; Îxin and Îxout, are the fractional voltage drops on internal and external ion entry, respectively; Îxpore is fractional voltage drops between two K+ binding sites within the pore; Ïout and Ïin are the external and internal surface charge densities, respectively. f is the position of the transition barriers. See for detailed description of model parameters.
	Figure 7. . A molecular and functional model of the pore region. (A) Simple estimate of the surface charge density in hSlo by considering the area delimited by the circle (25 Ã diameter). A surface charge density of â¼0.008 eâ/Ã2 can be estimated for the area including the four negative charges of the D292 residues. This value is very close to the fitted value of 0.0077 eâ/Ã2. (B) Cartoon representation of the proposed role of D292 as a site for accumulating K+ ions in the outer vestibule. The selectivity filter is depicted with five K+ binding sites. The reduced [K+] in the outer vestibule of hSlo-D292N results in reduced accessibility of K+ to the outer binding site of the selectivity filter. Inner negative charge residues participating in ion conduction are shown at the bottom of the inner vestibule (Brelidze et al., 2003; Nimigean et al., 2003).

References [+] :

Adelman, Calcium-activated potassium channels expressed from cloned complementary DNAs. 1992, Pubmed, Xenbase