Zhang Y et al. (2006), Ring of negative charge in BK channels facilita...

XB-ART-34498

J Gen Physiol 2006 Aug 01;1282:185-202. doi: 10.1085/jgp.200609493.

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Ring of negative charge in BK channels facilitates block by intracellular Mg2+ and polyamines through electrostatics.

Zhang Y , Niu X , Brelidze TI , Magleby KL .

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Species referenced: Xenopus laevis
Genes referenced: tbx2 tes

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	Figure 1. Intracellular Mg2+ blocks both WT and mutant (E321N/E324N) channels in a voltage- and concentration-dependent manner. A and B present ribbon structures of MthK for a side view of two opposed subunits and for a bottom view looking from the cytoplasm toward the channel for all four subunits, obtained with Swiss Protein Viewer and coordinates from Jiang et al. (2002a). The negatively charged residues E321 and E324 in BK channels are projected onto the ribbon structures as space filling structures (red and blue), which are substituted for E92 and L95 in MthK. Brelidze et al. (2003) substituted for R93 and E96 in their Fig. 1. It is unclear which substitution would be closer to the unknown structure of the BK channel. (C) Representative single-channel currents recorded from WT (top traces) and E321N/E324N (lower traces) BK channels expressed in oocytes with symmetrical 150 mM KCl and the indicated Mg2+i concentration. Membrane potential: +100 mV. Effective filtering: 5 kHz. Channel opening indicated by upward going currents. (D and E) Plots of outward unitary current amplitude versus voltage at the indicated Mg2+ from WT (filled symbols) and mutant (open symbols) channels, respectively. The intracellular solutions for all figures before adding the blocking ions or increasing intracellular KCl were (in mM) 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, pH 7.0. The lines are descriptions with Eq. 5 for simultaneous fitting of the data in D for WT channels: KBap(0) for Mg2+i = 48.3 ± 3.0 mM, d = 0.25 ± 0.01, and n = 0.64 ± 0.01, and simultaneous fitting of the data in E for E321N/E324N channels: KBap(0) for Mg2+i = 143 ± 16.6 mM, d = 0.19 ± 0.01, and n = 0.61 ± 0.02.
	Figure 2. The ring of charge facilitates Mg2+i block through an electrostatic mechanism. (A) The ring of negative charge increases the Mg2+i block for 150 mM K+i at all examined voltages. Plots of the ratio of unitary current with and without Mg2+i (iMg2+/i0) versus voltage from both WT (filled symbols) and E321N/E324N (open symbols) channels. The parameters for the lines calculated with Eq. 5 are the same as in Fig. 1. (B) The ring of negative charge no longer increases Mg2+i block when K+i is increased to 3 M. Plots of unitary current for WT channels (filled symbols) and mutant channels (open symbols) with intracellular KCl increased to 3 M. The WT symbols were shifted right by 5 mV so that they can be seen. The lines are fits from Eq. 5 with KBap(0) for Mg2+i = 561 ± 12 mM, d = 0.19 ± 0.01, and n = 1.01 ± 0.05 for both WT and E321N/E324N channels. (C) Increasing K+i decreases Mg2+ block. Plots of the ratio of unitary current from E321N/E324N channels with and without 20 mM Mg2+i (iMg2+/i0) versus voltage for three different K+i. The parameters for the fits with 150 and 3 M K+i are given in Figs. 1 and 2, respectively. The parameters for 500 mM K+i are KBap(0) = 492 ± 32 mM, d = 0.19 ± 0.01, and n = 0.85 ± 0.04.
	Figure 3. The ring of negative charge decreases both the apparent KB and increases the voltage dependence of Mg2+i block. (A) Removing the ring of charge increases the KBap for Mg2+i block. Plots of ratio of current amplitude at +100 mV with and without Mg2+i (iMg2+/i0) over a range of Mg2+i in WT (filled circles) and E321N/E324N (open circles) channels. The lines are fits with the Hill equation (Eq. 1): WT channels, KBap (+100 mV) for Mg2+i = 6.4 mM, n = 0.64 (continuous lines); E321N/E324N channels, KBap (+100 mV) = 33.8 mM, n = 0.65 (dashed lines). (B) Removing the ring of charge increases KBap. Semilogarithmic plots of KBap against voltage for both WT and E321N/E324N channels. The lines are the fits with Eqs. 2 and 3 with projected KBap (0 mV) for Mg2+i block of 42.3 mM for WT and 89.1 mM for E321N/E324N. (C and D) The Woodhull equation with added (negative) cooperativity accounts for Mg2+i block in both WT and E321N/E324N channels. Plots of iMg2+/i0 over a range of voltages at the indicated Mg2+i for WT (filled symbols) and E321N/E324N (open symbols) channels. The continuous lines are from simultaneously fitting the WT data in C and the dashed lines are from simultaneously fitting the E321N/E324N data in D. The parameters for the fitting are given in Fig. 1. (E and F) Same as C and D, except that n in Eq. 5 was set to 1.0 so that there was no cooperativity. The Woodhull equation without negative cooperativity could not simultaneously describe the data obtained over a range of concentrations of Mg2+i.
	Figure 4. Natural polyamines block WT BK channels in a concentration- and voltage-dependent manner, with the blocking sequence: spermine > spermidine > putrescine. (A) Representative single-channel currents at +140 mV with the different polyamines at the indicated concentrations. (B) Concentration and voltage dependence of spermine block. Plots of unitary current versus voltage at the indicated concentrations of spermine. The lines are simultaneous fits with Eq. 5 with KBap(0) = 8.0 ± 1.0 mM, d = 0.19 ± 0.01, n = 0.54 ± 0.02. (C) Comparison of block by spermine, spermidine, and putrescine. Plots of unitary current versus voltage for the indicated natural polyamines at 1 mM. The fitted parameters for each polyamine were determined by simultaneously fitting data obtained over a range of concentrations for each polyamine, as in B. Only the data at 1 mM are presented. The data for spermine are from B. The lines are separate fits with Eq. 5 for spermidine: KBap(0) = 38.4 ± 3.3 mM, d = 0.25 ± 0.03, n = 0.75 ± 0.04, and for putrescine: KBap(0) = 180 ± 17 mM, d = 0.33 ± 0.01, n = 0.85 ± 0.04. (D) Natural polyamines at 1 mM do not block inward currents through BK channels. Plots of inward unitary currents vs. voltage with and without 1 mM polyamines. (E) Structures of the natural polyamines.
	Figure 5. The ring of negative charge is a major site contributing to polyamine block, with block decreasing as the net charge in the ring of charge is decreased. (A) Representative single-channel currents with and without 1 mM spermine at +140 mV from channels with different net charge in the ring of charge: WT (−8), E321N (−4), E324N (−4), and E321N/E324N (0). The net charge in the ring of charge also changes the unitary current amplitude (Brelidze et al., 2003) so block must be judged by the ratio of unitary currents in the lower panel to those in the upper panel. (B) Removing the ring of negative charge greatly decreases spermine block. (Compare to Fig. 4 B). Plots of unitary currents versus voltage for E321N/E324N channels for the indicated spermine concentrations. The lines are simultaneous fits from Eq. 5 with KBap(0) = 721 ± 9 mM, d = 0.19 ± 0.02, n = 0.53 ± 0.07. (C) The degree of natural polyamine block is proportional to the net charge of the ring of charge. Plots of the ratio of unitary current with and without 1 mM spermine versus the net charge on the ring of charge for putrescine, spermidine, and spermine. For comparison, the ratio of unitary currents with and without 1 mM Mg2+i is also shown (filled circles). The overlapping points for a net charge of −4 indicate that E321 and E324 have equivalent effects on the block. (D and E) The Woodhull equation with added (negative) cooperativity accounts for spermine block in both WT and E321N/E324N channels. Plots of ispermine/i0 over a range of voltage at the indicated spermine for WT (filled symbols) and E321N/E324N (open symbols) channels. The continuous lines are from simultaneously fitting the WT data in D, and the dashed lines are from simultaneously fitting the E321N/E324N data in E. The parameters for the fitting are given in Fig. 4 B and Fig. 5 B.
	Figure 6. The ring of negative charge facilitates polyamine block through an electrostatic mechanism. (A and B) Plots of unitary current versus voltage with and without 1 mM spermine in the presence of 3 M intracellular KCl for WT and E321N/E324N channels. With 3 M K+i, the ring of charge has no effect on either spermine block or on the unitary current amplitude.
	Figure 7. The ring of negative charge increases the inward rectification of BK channels for cell-attached patches of membrane. (A and B) Representative single-channel currents recorded from WT and E321N/E324N channels for on-cell recording from Xenopus oocytes (top traces) and after excising the patch (bottom traces). Membrane potential: +140 mV. The oocytes were bathed in 150 mM KCl and 5 mM TES (pH 7) before forming a patch so that the membrane potential would be close to zero. (C and D) Plots of unitary current versus voltage from cell-attached (open symbols) and excised patches (filled symbols) from WT and E321N/E324N channels, respectively. Excised patch data are the same as in Fig. 1 (D and E). Three representative on-cell recordings, each from a different patch, are shown for WT and E321N/E324N channels. Note the variability in response between on-cell patches from different oocytes. The lines have no theoretical meaning. Filtering was 10 kHz in A and 5 kHz in B.
	Figure 8. Schematic diagram of a possible mechanism for intracellular Mg2+ and polyamine block in BK channels. The ring of negative charge attracts Mg2+ and polyamines, which then displaces K+ from the entrance to the inner vestibule. Mg2+ and polyamine also enter further into the inner vestibule, attracted by the dipole formed by the pore helixes, where they can displace K+ both physically and electrostatically from the inner vestibule and also from the entrance to the selectivity filter.

References [+] :

Armstrong, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. 1971, Pubmed