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Figure 1. The W4E mutation renders β2-mediated BK current inactivation properties similar to those of β3b. (A) Currents from a patch expressing mSlo1+β3b channels in response to a family of depolarizations. Inset shows the same currents on a time scale equivalent to C and E. (B) Normalized conductance data from mSlo1+β3b channels. Peak (â¡), steady-state (â¯), and tail (â³) currents were measured. Peak currents â¤40 mV were fit with a Boltzmann equation, and all current values were normalized to the positive asymptote of this fit (n = 8). (C) Current arising from a patch expressing mSlo1+β2 channels. (D) Normalized peak conductance data from mSlo1+β2. (n = 11) (E) Current arising from a patch expressing mSlo1+β2-W4E channels. (F) Normalized peak, steady-state, and tail current values calculated as for B (n = 10).
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Figure 2. β2-W4E channels redistribute extremely rapidly between inactive and conducting conformations. Channels expressing mSlo1+β2 (A) or mSlo1+β2-W4E (B) were depolarized for 1 ms (black) or 40 ms (red) and allowed to recover as shown. After 40-ms depolarizations, mSlo1+β2-W4E displayed an approximately twofold increase in conductance associated with repolarization (*). (C) Fractional change in instantaneous tail current conductance at â80 mV compared with conductance at +160 mV, immediately before repolarization (β2, n = 6; β2-W4E, n = 3; error bars, ±SEM). (D) A patch expressing mSlo1+β2-W4E was subjected to the indicated voltage protocol. Again, tail current conductance is increased immediately after a long depolarizing prepulse; arrows indicate the instantaneous tail current value during the brief repolarization. This increase is reversed immediately after the onset of a second depolarizing pulse.
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Figure 3. Inactivation of β2-W4E is associated with biphasic tail currents. (A) mSlo1+β2-W4E tail currents at 40 mV increments, after a 200-ms depolarization as shown. (B) mSlo1+β2-W4E tail currents at 40-mV increments, after a 5-ms depolarization as shown. Insets show tail currents on an expanded time base. (C) Time constants of exponential fits to tail currents from mSlo1+β2-W4E. Tail currents after short depolarizations were fit with single exponentials, and tail currents after long depolarizations were fit with the sums of two exponentials. â¯, decaying phase after long depolarization; â¡, rising phase after long depolarization; â³, decaying phase after short depolarization (n = 4).
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Figure 4. Instantaneous currentâvoltage relationships change with activation step duration. This change requires that channels occupy multiple distinct open states, one of which is in rapid equilibrium with an inactivated state. (A) Patches expressing mSlo1+β2 channels were activated with the indicated voltage protocol, with a depolarizing prepulse lasting 0.5 ms (top traces), 2 ms (middle traces), and 10 ms (bottom traces). For display purposes, raw currents are shown at 20-mV increments. Current was measured 120 μs after the onset of the voltage step from the prepulse potential, after settling of the capacitance transient. For each protocol, these currents were normalized to the value recorded at â100 mV and plotted as follows: â¯, 0.5 ms; â¡, 2 ms; â³, 10 ms (n = 6). (B) Patches expressing mSlo1+β2-W4E were recorded as in A (n = 7). (C) Patches expressing mSlo1+β2-W4E were treated with trypsin (1 mg/ml) at the cytoplasmic face until modification of macroscopic current was complete. Trypsin-modified patches were then recorded as above (n = 4).
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Figure 5. mSlo1+β2-W4E channels exhibit short reopenings with characteristics expected for the preinactivated O* state. (A) Recordings from a patch containing one mSlo1+β2-W4E channel using the voltage protocol shown. Bottom trace shows an ensemble average of 40 consecutive sweeps. (B) Histogram of open-time durations from this patch, with bins distributed logarithmically. At event durations less than twice the filter rise time (2Tr = 66 μs), significant numbers of events are missed during a half-amplitude idealization. Bins corresponding to this range of times are therefore underestimates and are shown in lighter gray. Line is a three-term exponential fit to these data, with Ïslow = 2.3 ms. (C) Recordings from the same patch after treatment with trypsin (1 mg/ml à 2 min) to the intracellular face. Bottom trace shows an ensemble average of 100 consecutive sweeps. (D) Histogram of open-time durations after trypsin treatment, with bins distributed logarithmically. Shading as in B. Line is a one-term exponential fit to these data, with Ï = 1.6 ms.
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Figure 6. Wild-type channels undergo occasional short-lived reopenings. (A) Recordings from a patch containing one mSlo1+β2 channel using the voltage protocol shown. A single brief reopening is shown (*). (B) Recording from a patch containing at least 15 mSlo1+β2 channels during a lengthy depolarization to +80 mV. Insets 1â3 show segments of this trace on an expanded baseline. (C) Histogram of open-time durations from this patch, with bins distributed logarithmically. Events less than twice the filter rise time (2Tr = 66 μs) are shown in lighter gray. Line shows a fit to the data with a double exponential decay. Only events >2Tr were included in the fit. Ïslow = 3.0 ms.
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Figure 7. Channels arrive at initial long- and short-lived open-ings by different kinetic paths. (A) Histograms of open-time duration for all openings (solid bars) and initial openings (open bars) of a single mSlo1+β2-W4E channel after depolarizations to +80 mV. Bins are spaced logarithmically. (B) Histograms of the latency before initial opening, binned linearly and segregated by the duration of this first opening. Initial openings were categorized into two groups, those lasting >0.2 ms and those <0.2 ms, based on the nadir between the two components of the open-time histograms shown in A. Lines show single-exponential fits to the decaying phases of the latency histograms (latencies â¥2 ms). For openings >0.2 ms (black), Ï = 2.2 ms; for openings â¤0.2 ms (red), Ï = 4.0 ms. (C) Scatterplot displaying the durations of initial openings versus the durations of the latency before opening. Events were again segregated as âshortâ (red) or âlongâ (black) by a criterion of 0.2 ms. Lines show the average duration and latency (computed logarithmically) for each population. For long events, the average latency was 2.31 ms (mean ± SEM, 2.25â2.38 ms, n = 1022). For short events, the average latency was 3.86 ms (mean ± SEM, 3.68â4.05 ms, n = 558).
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Figure 8. Models of preinactivation. (A) A six-state Markov scheme incorporating Scheme 3 with the addition of an extra, nonconducting resting state, CR. In the absence of allosteric coupling, rate constants on opposite sides of the central loop are assumed to be identical (e.g., kCâC* = kOâO*) To simulate removal of inactivation by trypsin, all states below the dotted line were removed. (B) Predictions of the scheme shown in A, with rate constants as listed in Table I. Rate constants were estimated from data acquired at +80 mV and 10 μM Ca2+. Top two traces, simulated single channel data, superimposed on 1 pA RMS noise, digitally lowpass filtered at 10 kHz. Bottom trace, simulated macroscopic current at +80 mV. Dotted line is zero-current level. (C) Simulated single-channel data after removal of inactivation by âtrypsin,â processed as above. (D) A structural conception of the preinactivated state. Images depict a cartoon showing two opposing α subunits from the mSlo1 tetramer, flanked by two β2 subunits. The five states shown correspond to those in Scheme 3. β, β2 subunit; OH, outer helix bundle (S0âS4); IH, inner helix bundle (S5âS6); T1, channel cytoplasmic domain (see Long et al., 2005). The depolarization-induced change permissive for preinactivation is represented as movement at the cytoplasmic face of the outer helix bundle. Possible locations for the interaction associated with preinactivation are shown in green in the C* and O* states.
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