Moss BL, Magleby KL.

AR32805 NIAMS NIH HHS , R01 AR032805 NIAMS NIH HHS

	Figure 1. Schematic diagram of wild-type mSlo1 channels, wild-type mSlo3 channels, mSlo1 tail channels and mSlo3 tail channels. The position of the calcium bowl in the mSlo1 tail is indicated. The proposed membrane topology of the four studied channels is shown in the top diagram and the components of the various channels is indicated in the schematic diagrams below. The mSlo1 tail channels are expressed from mSlo1 core domains (S0–S8) and mSlo1 tail domains (S9–S10). The mSlo3 tail channels are expressed from mSlo1core domains and mSlo3 tail domains. The mSlo3 core domain (residues 35–641) shares 56% identity with the mSlo1 core domain. The mSlo3 tail domain (residues 686–1136) shares 39% identity with the mSlo1 tail domain (Schreiber et al. 1998). A linker region with little identity is found between S8 and S9. Compared with the mSlo1 tail, the mSlo3 tail has six fewer negatively charged residues in the calcium bowl region. (Adapted from Schreiber et al. 1999.)
	Figure 2. Wild-type mSlo1 channels and wild-type mSlo3 channels have different single-channel kinetics. (A) Single-channel current recordings from wild-type mSlo1 and wild-type mSlo3. Upward (outward) currents indicate channel opening. The mSlo1 patch contained three channels, with a mean PO = 0.040. The mSlo3 patch contained a single channel with a PO = 0.047. Ca2+i = 0 μM, pH 7.4. Membrane potential = +80 mV. Currents were low-pass filtered at 8 kHz. (B) Representative bursts of openings from A on a faster time base. (C–F) Open and closed 1-D dwell-time distributions for mSlo1 (C and D) and mSlo3 (E and F). The distributions have been scaled to contain the same number of intervals (100,000) to facilitate comparison. The dashed line in D indicates the duration of gaps between bursts after correcting for the number of channels in the patch (materials and methods). The arrows indicate the mean open times and mean durations of the gaps between bursts. The continuous lines are fits with sums of exponential components with the following time constants and areas. (C, open) 0.07 ms, 0.26; 0.28 ms, 0.54; 0.73 ms, 0.20. (D, closed) 0.06 ms, 0.46; 0.21 ms, 0.09; 3.30 ms, 0.17; 7.22 ms, 0.28. (E, open) 0.04 ms, 0.34; 0.10 ms, 0.66. (F, closed) 0.02 ms, 0.25; 0.14 ms, 0.38; 1.93 ms, 0.23; 7.58 ms, 0.14.
	Figure 3. Bursting kinetics for wild-type mSlo1 channels differ from wild-type mSlo3 channels at comparable PO's. (A–D) Plots of the indicated mean bursting parameters for each channel type. Compared with mSlo1 channels, mSlo3 channels showed a briefer mean burst duration (P < 0.01), a briefer mean open time (P < 0.001), and a briefer mean gap duration between bursts (P < 0.02), while showing no significant difference in the mean number of openings per burst (P = 0.5). Error bars represent SEM. Data were obtained from five patches containing mSlo1 channels and five patches containing mSlo3 channels. Mean PO: mSlo1 = 0.030 ± 0.005 (range: 0.014–0.040); mSlo3 = 0.030 ± 0.009 (range: 0.011–0.055). All data were obtained at a Ca2+i of 0 μM and a membrane potential of +80 mV.
	Figure 4. Replacing the mSlo1 tail with the mSlo3 tail greatly decreases Ca2+ sensitivity and increases activity in the absence of Ca2+. (A) Single-channel currents recorded from a single mSlo1 tail channel and a single mSlo3 tail channel at different Ca2+i. Membrane potential: +30 mV. Currents were low-pass filtered at 8 kHz. (B and C) Plots of PO versus Ca2+i for four mSlo1 tail channels (B) and seven mSlo3 tail channels (C). The lines are fits of the Hill equation, with the mean values of the parameters given in the text. The mSlo1 tail and mSlo3 tail channels in A are represented by closed and open triangles, respectively, in B and C.
	Figure 5. Replacing the mSlo1 tail with the mSlo3 tail decreases the Ca2+ sensitivity of both mean open and mean closed times. Mean open time and mean closed time versus Ca2+i for four mSlo1 tail channels (A and C) and four mSlo3 tail channels (B and D). Symbols correspond to those in Fig. 4. Membrane potential: +30 mV. Currents were low-pass filtered at 8 kHz.
	Figure 6. Replacing the mSlo1 tail with the mSlo3 tail decreases the voltage dependence. (A) Single-channel recordings from patches containing a single mSlo1 tail channel and a single mSlo3 tail channel at the indicated membrane potentials. The records were low-pass filtered at 3 kHz. (B and C) Plots of PO versus membrane potential for five mSlo1 tail channels (B) and five mSlo3 tail channels (C). The lines are fits of the Boltzmann equation, with the mean values of the parameters given in the text. For ease of comparison, the dotted lines in B plot the fits to the mSlo3 tail channels in C. The mSlo1 tail channels were studied in 0 μM (closed circle, closed diamond), 8.7 μM (closed inverted triangle and closed square) and 25.6 μM (closed triangle) Ca2+i. The mSlo3 tail channels were studied in 0 μM (open triangle, open square, and open circle), 8.7 μM (open diamond), and 15.2 μM (open hexagon) Ca2+i. The records shown in A are represented by inverted closed triangles in B for the mSlo1 tail channel and by upright open triangles in C for the mSlo3 tail channel.
	Figure 7. Replacing the mSlo1 tail with the mSlo3 tail decreases burst duration. (A) Single-channel current recordings from patches containing a single mSlo1 tail channel and a single mSlo3 tail channel. For the mSlo1 tail channel, Ca2+i = 15.2 μM and PO = 0.30. For the mSlo3 tail channel, Ca2+i = 0 μM and PO = 0.34. Membrane potential: +30 mV. Both recordings were low-pass filtered at 8 kHz. (B) Representative bursts of openings from A on a faster time base. (C–F) Open and closed 1-D dwell-time distributions for the mSlo1 tail channel (C and D) and mSlo3 tail channel (E and F) shown in A and B. The distributions have been scaled to contain the same number of intervals (100,000) to facilitate comparison. The arrows indicate the mean open times and the mean durations of the gaps between bursts. The time constants and areas of the exponential components are as follows. (C, open) 0.05 ms, 0.16; 0.27 ms, 0.20; 1.08 ms, 0.39; 2.57 ms, 0.25. (D, closed) 0.03 ms, 0.44; 0.11 ms, 0.21; 0.35 ms, 0.15; 1.17 ms, 0.08; 6.15 ms, 0.08; 22.94 ms, 0.04. (E, open) 0.06 ms, 0.23; 0.56 ms, 0.45; 1.49 ms, 0.32. (F, closed) 0.03 ms, 0.40; 0.19 ms, 0.32; 1.10 ms, 0.13; 5.74 ms, 0.15.
	Figure 8. Bursting kinetics for mSlo1 tail channels differ from mSlo3 tail channels at the same PO. (A–D) Plots of indicated bursting parameters versus PO. Compared with mSlo1 tail channels, mSlo3 tail channels showed a briefer mean burst duration (A), a briefer mean open time (B), a decrease in the mean number of openings per burst (C), and briefer durations of gaps between bursts (D). For both mSlo1 tail channels and mSlo3 tail channels, each plot contains estimates from 10 datasets from three different channels. Membrane potential: +30 mV. All recordings were low-pass filtered at 8 kHz. Each symbol type plots data from a different channel. The channels shown in Fig. 7 (A and B) are represented by a closed square for the mSlo1 tail channel and an open triangle for the mSlo3 tail channel. For comparison, each plot includes the mean burst parameters for the wild-type mSlo1 channels (closed circle with crosshair) and wild-type mSlo3 channels (open circle with crosshair) from Fig. 3.
	Figure 9. Replacing the mSlo1 tail with the mSlo3 tail does not change the number of detected kinetic states entered during gating. Estimates of the number of detected open (A) and closed (B) states entered during gating plotted against the number of intervals analyzed. Estimates plot the number of significant exponential components required to describe the 1-D dwell-time distributions. For each channel type, the 15 estimates of the open states and 15 estimates of the closed states are from fitting 15 different datasets from five different single-channel patches. Multiple datasets were obtained from each channel by obtaining data at different Ca2+i, which ranged from 0 to 67 μM. Membrane potential: +30 mV. All recordings were low-pass filtered at 8 kHz.
	Figure 10. A comparison of 2-D dwell-time distributions and dependency plots obtained for mSlo1 tail channels and mSlo3 tail channels at similar PO's indicates that the two channel types have distinct kinetics, yet similar kinetic structures. Compare A with C and E with G to see differences in the 2-D dwell-time distributions between mSlo1 tail channels and mSlo3 tail channels at comparable PO's. The arrows in C and G indicate the higher occurrence of brief open intervals adjacent to brief closed intervals observed in mSlo3 tail channels. Compare B with D and F with H to see the similar saddle shape of the dependency plots between mSlo1 tail channels and mSlo3 tail channels. The heavy lines in the dependency plots indicate a dependency of zero. A–D present the 2-D dwell-time distributions and dependency plots for the mSlo1 tail channel and the mSlo3 tail channel shown in Fig. 7.
	Figure 11. Replacing the mSlo1 tail with the mSlo3 tail decreases single-channel conductance. (A) Single-channel current recordings from a single mSlo1 tail channel and a single mSlo3 tail channel. The dashed lines indicate the maximum current amplitude observed for each channel type. Membrane potential: +70 mV. Records were low-pass filtered at 5 kHz. (B) Representative bursts from A on a faster time base. (C) Plots of single-channel current amplitude versus membrane potential for the channels shown in A and B. The slope conductances were 285 pS for the mSlo1 tail channel and 243 pS for the mSlo3 tail channel. (D) Histograms of slope conductance measurements from 20 patches containing mSlo1 tail channels and 24 patches containing mSlo3 tail channels. For each plot, the superimposed line represents the fit of a Gaussian function to the histogram, yielding mean slope conductances of 276 ± 4 pS and 238 ± 4 pS for the mSlo1 tail channels and mSlo3 tail channels, respectively.
	Figure 12. Replacing the mSlo1 tail with the mSlo3 tail increases sensitivity to block by internal TEA. (A) Single-channel current recordings from patches containing a single mSlo1 tail channel and a single mSlo3 tail channel at the indicated concentrations of internal TEA (TEAi). Membrane potential: +70 mV. Recordings were low-pass filtered at 5 kHz. (B and C) Plots of single-channel current amplitude versus membrane potential at the indicated TEAi for the mSlo1 tail channel (B) and mSlo3 tail channel (C) shown in A. (D) Plot of single-channel current amplitude as a percent maximum current versus TEAi for the channels shown in A–C, but at a membrane potential of +90 mV. The solid lines are fits of a Langmuir function () which yielded Kd values (indicated by dotted lines) of 77 mM and 55 mM for the mSlo1 tail channel and mSlo3 tail channel, respectively. Kd values at 0 mV were estimated using the Woodhull 1973 equation (). The inset in D plots ln[(gO/gB) − 1] against membrane potential, where gO and gB represent the single-channel conductance in the absence and presence of 100 mM TEA, respectively. Analysis of the y-intercepts yielded Kd values of 207 and 137 mM at 0 mV for the mSlo1 tail channel and mSlo3 tail channel, respectively. (E) Mean Kd (±SEM) values plotted against membrane potential for mSlo1 tail channels (two experiments) and mSlo3 tail channels (three experiments). The lines are regression fits to show the trend in the data.

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