Zhou Y et al. (2010), Glycine311, a determinant of paxilline block in...

XB-ART-41841

J Gen Physiol 2010 May 01;1355:481-94. doi: 10.1085/jgp.201010403.

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Glycine311, a determinant of paxilline block in BK channels: a novel bend in the BK S6 helix.

Zhou Y , Tang QY , Xia XM , Lingle CJ .

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The tremorogenic fungal metabolite, paxilline, is widely used as a potent and relatively specific blocker of Ca(2+)- and voltage-activated Slo1 (or BK) K(+) channels. The pH-regulated Slo3 K(+) channel, a Slo1 homologue, is resistant to blockade by paxilline. Taking advantage of the marked differences in paxilline sensitivity and the homology between subunits, we have examined the paxilline sensitivity of a set of chimeric Slo1/Slo3 subunits. Paxilline sensitivity is associated with elements of the S5-P loop-S6 module of the Slo1 channel. Replacement of the Slo1 S5 segment or the second half of the P loop results in modest changes in paxilline sensitivity. Replacing the Slo1 S6 segment with the Slo3 sequence abolishes paxilline sensitivity. An increase in paxilline affinity and changes in block kinetics also result from replacing the first part of the Slo1 P loop, the so-called turret, with Slo3 sequence. The Slo1 and Slo3 S6 segments differ at 10 residues. Slo1-G311S was found to markedly reduce paxilline block. In constructs with a Slo3 S6 segment, S300G restored paxilline block, but most effectively when paired with a Slo1 P loop. Other S6 residues differing between Slo1 and Slo3 had little influence on paxilline block. The involvement of Slo1 G311 in paxilline sensitivity suggests that paxilline may occupy a position within the central cavity or access its blocking position through the central cavity. To explain the differences in paxilline sensitivity between Slo1 and Slo3, we propose that the G311/S300 position in Slo1 and Slo3 underlies a structural difference between subunits in the bend of S6, which influences the occupancy by paxilline.

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Species referenced: Xenopus laevis
Genes referenced: kcna2 kcnma1 tbx2

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	Figure 1. Paxilline blocks Slo1, but not Slo3 current. (A) Traces show block of Slo1 by paxilline, and the general topology of the Slo1 α subunit is shown on the top. Currents were activated by a step to +120 mV in an inside-out patch exposed to 10 µM Ca2+ with the indicated concentrations of paxilline. The holding potential was 0 mV, but the command steps were preceded by a brief 10-ms step to −140 mV. (B) The time course of paxilline block and unblock of Slo1 current is illustrated. Steps were to +100 mV. (C) G-V curves were generated from steady-state current levels for recordings in the absence and presence of paxilline (n = 4–7 patches). For this set of data, paxilline was applied at increasing concentrations, and full washout was not usually achieved (see B). Red lines correspond to a fit of Eq. 1, with Kb = 10.6 ± 0.49 nM with zc = zo = 0 e. (D) 500 nM paxilline is without effect on MC13 currents activated at +200 mV with pH 8.5. The composition of the MC13 construct is schematized at the top with Slo3 sequence shown in red. (E) MC13 currents are unaltered by 500 nM paxilline during 6 min of application. (F) 500 nM paxilline has little effect on MC13 G-V curves.
	Figure 2. S5–pore loop–S6 segments of the BK channel and the relative size of the paxilline molecule. (A) A homology model of the BK channel S5–pore loop–S6 structure was generated based on alignment with Kv1.2 and the available Kv1.2 crystal structure. Coloring identifies segments used for generation of chimeras as follows: red, S5; yellow, first half of pore loop; blue, second half of pore loop, including so-called turret; gray, S6. Orange residues are those that influence iberiotoxin binding (Giangiacomo et al., 2008). G311 in S6 is highlighted in magenta. A space-filling model of paxilline (to the same scale) is positioned adjacent to an S5 helix, but this positioning is not meant to imply anything about the position of paxilline in a blocking site. Note that the actual topology of the BK turret is entirely unknown. Furthermore, this cartoon does not take into account the likely large width of the BK central cavity. (B) The chemical structure of paxilline is shown.
	Figure 3. Replacement of the Slo1 S6 segment with the Slo3 sequence abolishes sensitivity to paxilline. (A) Replacing the Slo1 S5–P loop–S6 segment with the homologous Slo3 sequence (chimera MC2) abolishes paxilline sensitivity. The MC2 construct is diagrammed at the top. Traces show MC2 currents activated with 10 µM Ca2+ at +200 mV with and without 500 nM paxilline. (B) Paxilline has no effect on MC2 G-V curves (n = 4 patches). (C) Paxilline sensitivity persists after replacement of the Slo1 S5 segment with the homologous Slo3 sequence. The MC28 construct is diagrammed at the top. Traces show MC28 currents activated with 10 µM Ca2+ at +180 mV with and without paxilline. (D) G-V curves for MC28 with and without 50 nM paxilline (n = 3 patches) are shown. Red lines correspond to a fit of Eq. 1, with Kbc = Kbo = 30.4 ± 1.7 nM with no voltage dependence. (E) Paxilline sensitivity is lost when the Slo1 S6 segment is replaced with the homologous Slo3 sequence. The MC10 construct is diagrammed at the top. Traces show MC10 currents activated with 0 Ca2+ at +200 mV with and without paxilline. MC10 is strongly activated by Ca2+, but like Slo3 and MC13, it is also strongly blocked by µM Ca2+. (F) G-V curves are shown for MC10 with and without paxilline (n = 4 patches).
	Figure 4. The P loop segment modestly influences paxilline sensitivity. (A) Exchanging the Slo1 P loop with the homologous Slo3 sequence (MC6) on paxilline sensitivity has little or weak effects on paxilline sensitivity. The MC6 construct is schematized on the top. Traces show the effect of various paxilline concentrations on MC6 currents evoked by a step to +200 mV with 10 µM Ca2+. (B) G-V curves are plotted for MC6 with and without paxilline (n = 4–5 patches). The red lines correspond to the best fit of a model in which paxilline blocks both open and closed channels identically (Kb = 23.2 ± 1.0 nM) with no voltage dependence. (C) Replacing the second half of the Slo1 P loop with the homologous Slo3 sequence (MC18) has little effect paxilline sensitivity. The MC18 construct is diagrammed at the top. Traces show MC18 currents activated with 10 µM Ca2+ at +180 mV with and without paxilline. (D) G-V curves are plotted for MC18 with and without paxilline (n = 4 patches), with the red lines showing a fit of Eq. 2 with Kb = 30.0 ± 1.4 and z = 0 e. (E) Replacing the first half of the Slo1 P loop with the homologous Slo3 sequence (MC8) increases paxilline sensitivity. The MC8 construct is diagrammed at the top. Traces show MC8 currents activated with 10 µM Ca2+ at +200 mV in the presence and absence of paxilline. (F) G-V curves are displayed for MC8 with and without paxilline (n = 5 patches). Red lines correspond to a fit of Eq. 1 with equivalent open- and closed-channel block (Kb = 4.04 ± 0.10 nM) with no voltage dependence.
	Figure 5. Replacing the first half of the Slo1 P loop with the Slo3 sequence increases paxilline block affinity and speeds up paxilline block and unblock rates. (A) The time course of onset and recovery of paxilline block for Slo1 (black) and MC8 (red) is shown for representative inside-out patches for each construct. For MC8, 20 nM paxillline was applied (horizontal bar), and 100 nM was used for Slo1. Peak current activated at +200 mV was measured every 2 (Slo1) or 3 (MC8) seconds and plotted against time. (B) Time constants of onset of paxilline block (τon) and recovery (τoff) are plotted for Slo1 (at 100 nM) and MC8 (at 20 nM). Red dots are individual determinations, and bars indicate means and SEM. (C) The forward block rate and paxilline block affinity were calculated for each individual patch for Slo1 and MC8. Block rate (left axis) and affinity (right axis) were determined from τon = 1/([PAX]kf+ku), ku = 1/τoff, and Kb = ku/kf.
	Figure 6. Mutation of glycine 311 in Slo1 S6 abolishes high affinity block by paxilline. (A) A consensus alignment of Slo1 and Slo3 S6 segments is shown. Non-conserved residues are in red. (B) Paxilline is without effect on Slo1-G311S. Currents were activated with 10 µM Ca2+ at +200 mV, and 500 nM paxilline produces no effect. On the bottom, G-V curves for Slo1-G311S with and without 500 nM paxilline (n = 3 patches) are plotted. (C) Paxilline is without effects on Slo1-G311A. Currents were activated with 10 µM Ca2+ at +200 mV, and 500 nM paxilline had only a minor effect. G-V curves plotted on the bottom also show lack of paxilline effect (n = 5 patches). (D) Paxilline sensitivity persists in Slo1-A313I. Currents activated with 100 µM Ca2+ are blocked by 20 and 100 nM paxilline, as also shown in the G-V curves on the bottom (n = 5 patches). (E) Paxilline blocks Slo1-M314L. Currents activated with 100 µM Ca2+ are blocked in a concentration-dependent fashion by paxilline, as summarized in the G-V curves on the bottom (n = 8 patches). Red line corresponds to fit with equivalent voltage-independent block of open and closed states with Kb = 13.3 ± 0.80 nM.
	Figure 7. Paxilline sensitivity is restored in constructs with a Slo3 S6 by a glycine at position S300. (A) Paxilline blocks MC10-S300G. 50 nM paxilline markedly inhibits MC10-S300G currents activated at +150 mV with 0 Ca2+. (B) G-V curves are plotted for MC10-S300G with and without 50 nM paxilline (n = 4 patches). Red lines are a fit of Eq. 2 with Kb = 13.1 ± 0.6 nM and no voltage dependence. (C) Paxilline more weakly blocks MC13-S300G. Traces show MC13-S300G currents activated at pH 8.5 at +200 mV. Inhibition occurs with high concentrations of paxilline. (D) G-V curves are plotted for MC13-S300G with and without paxilline (n = 3 patches). Red lines are a fit of Eq. 2 with Kb = 170.2 ± 7.2 nM and no voltage dependence.
	Figure 8. Other tests of factors influencing paxilline block. (A) Paxilline blocks construct Slo1-G310C. Traces show reduction of Slo1-G310C currents by the indicated paxilline concentrations with 10 µM of cytosolic Ca2+ and steps to +200 mV. (B) G-V curves are shown for paxilline block of Slo1-G310C. Red line corresponds to fit of voltage-independent block of open and closed states with Kb = 34.6 ± 1.8 nM. (C) Paxilline blocks DN5. Traces show paxilline block of DN5 in which the Slo1 cytosolic structure is replaced by Slo3. Currents were activated with cytosolic pH 8.5 with steps to +280 mV. (D) G-V curves are shown for paxilline block of DN5. Red lines show a fit with Kb = 6.3 ± 0.3 nM with z = 0 e. (E) Paxilline block on MC10-S300G G-V curves is similar at either pH 7.0 (black) or pH 8.5 (red). In both cases, 20 nM paxilline produces similar block. (F) Points plot the peak current during the application of 100 nM paxilline applied to outside-out patches expressing either Slo1 (black) or Slo1-G311S (red). Currents were activated by steps to +200 mV with 10 µM of pipette Ca2+ with a holding potential of 0 mV.
	Figure 9. Block of BK channels by other fungal alkaloids is also abolished by the G311S mutation. (A) A Slo1 G-V curve for activation with 10 µM Ca2+ is plotted for currents measured in the absence and presence of 10 nM aflatrem (n = 4 patches). (B) The time course of block by 10 nM aflatrem is shown for one patch. (C) 10 nM aflatrem is without effect on the G-V elicited by 10 µM Ca2+ for the Slo1-G311S construct. (D) Over 4 min of application of 10 nM aflatrem produces no reduction in current of Slo1-G311S. (E) The effect of 10 nM penitrem A on the Slo1 G-V curve is plotted. (F) The time course of block of Slo1 by penitrem A is plotted. (G) 10 nM penitrem A is without effect on Slo1-G311S conductance. (H) Over 5 min of application of 10 nM penitrem A has no effect on Slo1-G311S current. (I) Block of the Slo1 G-V by 10 nM verruculogen is plotted. (J) The time course of verruculogen block is displayed. (K) 10 nM verruculogen is without effect on Slo1-G311S conductance. (L) Over 5 min of verruculogen application is without effect on Slo1-G311S current.

References [+] :

Brelidze, Probing the geometry of the inner vestibule of BK channels with sugars. 2005, Pubmed, Xenbase