Sack JT , Aldrich RW , Gilly WF .

NS-17510 NINDS NIH HHS

	Figure 1. . Sidedness of BrMT effect on ShBΔ IK from excised oocyte patches. IK in A and B is from ShBΔ with C-type inactivation minimized by the T449Y mutation. The ShBΔ channels in C do not contain the mutation at position 449. (A) Effects of external BrMT on ShBΔ IK from an outside-out patch. BrMT greatly slows activation kinetics and leads to a reduction in peak amplitude. Voltage step to +20 mV from −100 mV. Thin trace: control condition. Thick trace: external solution with 5 μM BrMT. TCEP was not included in the internal solution. (B) BrMT applied to inside-out patches has no apparent effect on activation but reduces peak IK, induces a decline in amplitude after the peak, and slows deactivation kinetics. Step to +40 mV from −100 mV. Thin trace: control condition. Thick trace: internal solution with 2 μM BrMT. (C) The monomeric form of BrMT does not block ShBΔ channels from the internal side of an inside-out patch. Including the disulfide reducing agent TCEP (2 mM) with 10 μM BrMT eliminates effects of internal BrMT. Voltage step from −80 to +20 mV returning to −120 mV. Thin trace: control condition. Thick traces: internal solution includes either 10 μM BrMT and 2 mM TCEP or 10 μM BrMT but no TCEP.
	Figure 2. . Effects of 5 μM BrMT applied externally to ShBΔ channels. Data in all panels are from the same outside-out patch. (A) IK activation during voltage steps from −100 mV to a series of voltages. Thin traces: control condition. Thick traces: 5 μM BrMT. (B) IK deactivation after activating pulses to +20 mV. (C) Conductance-voltage relations from tail currents determined in the absence and presence of BrMT. Lines are fits of a fourth-power of a Boltzmann function (control: V1/2 = −59, z = 2.1; 5 μM BrMT: V1/2 = −3.4, z = 2.5). (D) BrMT does not affect rectification or reversal potential. Instantaneous I-V plot was constructed from tail current amplitudes after an activating pulse to +20 mV. Open circles: control condition. Filled circles: 5 μM BrMT. Lines are linear fits to the data. Reversal potentials from these fits are −58.2 mV (control) and −58.1 mV (BrMT). (E) Voltage dependence of activation and deactivation kinetics in the absence and presence of BrMT. Activation time constants were determined by a single exponential fit to the final 50% of the IK waveform. Deactivation time constants were determined by a single exponential fit to the entire decay of IK after an activating pulse to +20 mV. Open circles: τcontrol, control condition. Filled circles: τBrMT, 5 μM BrMT. Crosses: τBrMT/τcontrol.
	Figure 3. . Concentration dependence of external BrMT. Dose–response data from outside-out patches containing ShBΔ. (A) Thin trace: ShBΔ IK in response to a voltage step from −100 to +40 mV. Thick traces: IK at +40 mV in BrMT. (B) Slowing of IK activation by BrMT at +40 mV is plotted as the ratio of time constants from fits to activation kinetics (τBrMT/τcontrol). Different symbols represent values from different patches that underwent the entire dose–response. Crosses represent mean ± SE from 4–9 patches. (C) Effect of BrMT on peak IK amplitude. Peak IK was measured at +40 mV. Symbols correspond to same patches as in B. Crosses represent mean ± SE from 4–10 patches. (D) Conductance-voltage relations determined from different concentrations of BrMT applied to a single patch. Conductance values are from tail currents at −100 mV. Data at each concentration were normalized to the peak amplitude of a fourth-power Boltzmann fit (lines). Fits to BrMT data were constrained to have the steepness of the fit to control data (z = 3.4). V1/2 values from the fourth-power fits: control (open circles) = −49 mV; 1 μM (triangles) = −42 mV; 2 μM (inverted triangles) = −36 mV; 5 μM (filled circles) = −27 mV; 10 μM (diamonds) = −12 mV; 20 μM (squares) = −6 mV.
	Figure 4. . Effects of 5 μM BrMT on single ShBΔ channels. Analyses was conducted on IK from outside-out patches containing one functional ShBΔ channel during 100-ms steps to +40 mV from −100 mV. Data in A–G are from a single patch (patch 3 in H–L). In C–G, analyses of control currents are represented by thin traces, and IK in 5 μM BrMT by thick traces. In H–L, thick-rimmed grayed bars are measurements in BrMT and hollow bars under control conditions. (A) Representative traces containing single channel openings under control conditions. (B) Openings of the same channel in 5 μM BrMT. (C) Ensemble averages of sweeps containing channel openings (i.e., nonblank sweeps). (D) Cumulative first latencies from sweeps containing openings. (E) Unitary conductance does not change in BrMT. All-points histograms were compiled from sweeps without apparent substates. Bin width is 10 fA. (F) Logarithmically binned open durations in BrMT. Substates were not included in this analysis. Curve is fit for a single open state. The mean open time of 5.2 ms indicated by a vertical line. (G) Open durations under control conditions. Mean open time is 5.4 ms. (H) Time constants of exponential fits to the final 50% rise of cumulative first latencies. (J) Unitary conductance is unchanged by BrMT. Unitary IK in 5 μM BrMT versus control was 1.01 ± 0.02 (n = 6) while peak macroscopic IK was reduced to 0.80 ± 0.02 (n = 10). (K) Mean open time is little affected by BrMT (L) Conditional probabilities that a channel is open provided it has opened previously. Calculated for sweeps containing channel openings (see results and materials and methods). The first and last idealized events were not included in determination of pOpen.
	Figure 5. . Effects of BrMT on ShBΔ gating currents. Gating currents were recorded from CHO-K1 cells expressing ShBΔ channels. Similar effects on ShBΔ Ig were seen in nine cells. Thin traces: control condition. Thick traces: in 5 μM BrMT. The zero current baseline for gating charge integration (Q) was set individually for each trace, after the Ig transient decayed to steady-state. (A) IgON is slowed and peak amplitude is greatly reduced by BrMT. Voltage steps are to +20 mV from −100 mV. (B) Integrated charge movement (QON) from IgON in A. (C) IgOFF in BrMT is similar to control. Voltage steps are to −140 mV after a 200-ms activating pulse to +20 mV. (D) Integrated charge movement (QOFF) from IgOFF in C. QOFF in BrMT was 1.02 ± 0.02 that of control, n = 4 cells.
	Figure 6. . BrMT slows early but not late activating transitions of ShBΔ ILT. IK from ShBΔ V369I;I372L;S376T (ILT) in outside-out patches. HP = −60. Pipette solution did not contain TCEP. (A) Scheme depicting gating of ILT channels. (B) IK rise from ILT channels at +140 mV after 60-ms prepulses to either −140 or 0 mV. Note the slight delay apparent from −140 mV. (C) ILT activation during a step from −140 mV. IK in 5 μM BrMT is scaled to match peak control IK. Open circle: mean time to half maximal IK in 5 μM BrMT was 1.6 ± 0.2 times that of control, n = 5 patches. (D) ILT activation during a step from 0 mV. IK in 5 μM BrMT is scaled to match peak control IK. Note the near perfect IK overlay indicating that BrMT does not slow the late activation steps of the ILT channel. Open circle: mean time to half maximal IK in 5 μM BrMT was similar (1.1 ± 0.1 times control, n = 5 patches).
	SCHEME I.
	SCHEME II.
	SCHEME III.
	Figure 7. . Graded activation slowing can be modeled by rapid, reversible binding of BrMT to resting voltage sensors. (A) ShBΔ IK scaled to match peak current amplitudes. Thin trace: voltage step from −100 to +40 mV under control conditions. Thick traces: progressive slowing of activation at +40 mV by 1, 2, 5, 10, and 20 μM BrMT. (B) Predicted IK from an activation model with slow BrMT binding to each of four subunits that undergo a single activating transition. Subunits bound to BrMT activate 20-fold slower than those without. This value was chosen because the maximal slowing of ShBΔ activation seen was ∼20-fold in BrMT versus control (see D). The proportion of channels with 0, 1, 2, 3, or 4 BrMT bound was binomial. Thin line: control condition. Dotted line: saturating BrMT. Thick lines: [BrMT] = 0.2, 0.5, 1, 2, and 5× KD. (C) Predicted IK from Eq. 4. This equation represents an n4 model of activation with rapid BrMT binding as in Scheme IV. Increasing the concentration of BrMT slows activation by increasing the proportion of time subunits are bound to BrMT and thus unable to activate. Thin line: control condition. Thick lines: [BrMT] = 1, 2, 5, 10, and 20× KD. (D) Filled circles: ratio of τBrMT and τcontrol at +40 mV, n = 4–9. Line: fit of rapid binding model (Eq. 3) to data. KD from fit = 0.8 μM.
	SCHEME IV.

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