BK Channels Are Activated by Functional Coupling With L-Type Ca2+ Channels in Cricket Myocytes

Figures

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  Figure 1. Voltage-sensitive single K+ channel current in isolated cricket oviduct cells. (A) A single myocyte isolated from the lateral oviduct by optical microscopy. The scale bar is 50 μm. (B) Single K+ channel current from cell-attached patches under an extracellular K+ concentration ([K+]o) of 10 mM. Representative single-channel current traces at various holding potentials (Vpip) are indicated in the figure. c indicates a closed level. (C) Averaged single-channel current (i) -Vpip (i-Vpip) relationship (n = 6–14). Data points from +20 mV to −80 mV of Vpip were fitted by linear regression to obtain a slope conductance of 35.7 pS. (D) The average number of channel and single-channel open probability (NPo)–Vpip relationship of steady-state single-channel current recorded at each Vpip. The NPo–Vpip curve fits the Boltzmann function with the voltage for half-maximal activation of −38.9 mV and the slope of 12.3 mV. A total of 59 cells isolated from a total of 75 animals were used in the experiment and 65 tests were performed for data collection.

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  Figure 2. Single K+ channel IV relationship at various extracellular K+ concentrations. (A) Averaged IV data at various extracellular K+ concentrations ([K+]o), •, 140; ▲, 105; ◆, 70; ■, 35; ○, 10, from cell-attached patches. (B) The data show a semi-logarithmic plot of [K+]o against the difference in reversal potential obtained at 140 mM and the reversal potential obtained at each K+ concentration (Erev). The Erev was obtained by fitting the i–Vpip relationship of each K+ concentration in A by linear regression. The slope of the regression line shows 48.1 mV/decade. (C) The [K+]o dependence of slope conductance (g) is shown. The value of each conductance was obtained by fitting the i–Vpip relationship of each K+ concentration of A by linear regression. The solid curve fits the Michaelis–Menten equation. The inset shows the same data plotted in double reciprocal format. gmax, 117.6 pS; Km, 25.9 mM. A total of 56 cells isolated from a total of 46 animals were used in the experiment and 45 tests were performed for data collection.

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  Figure 3. Intracellular Ca2+ concentration dependence of K+ channel activation. (A) Representative traces of single K+ channel currents recorded under various intracellular Ca2+ concentration ([Ca2+]i) conditions at Vpip = 0 mV from excised inside-out patches. c and o indicate closed and open levels, respectively. (B) Average NPo-[Ca2+]i relationship of steady-state single-channel current. The data fit the Hill equation with an EC50 of 31.2 ± 10.6 nM and a Hill coefficient of 1.4. A total of 36 cells isolated from 32 animals were used in the experiment and 23 tests were performed to data collection.

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  Figure 4. Effect of tetraethylammonium and iberiotoxin on K+ channel current. (A) (top) Single-channel current in the absence (control) and presence of extracellular 0.5 mM tetraethylammonium (TEA) using the backfill method. (lower) Single-channel current in the absence (control) and presence of 10 nM extracellular iberiotoxin (IbTX) using the backfill method. The Vpip was 100 mV. c indicates a closed level. (B) The averaged amplitude of the single-channel currents in the absence and presence of TEA (t-value 25.16) or IbTX (t-value 25.16), respectively. *Significantly different (P < 0.05) from control values. (C) Average relative relationship in the presence of TEA concentration ([TEA]o) to the control value of single K+ channel current amplitude. The data fit the Hill equation with an IC50 of 318.8 ± 68.8 μM and a Hill coefficient of 1.0. A total of 83 cells isolated from 11 animals were used in the experiment, and 11 tests were performed for data collection.

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  Figure 5. Effect of Ca2+ removal from bath solution on BK channel activity. (A) Representative BK channel currents before (control) and after Ca2+ removal from bath solution (Ca2+-free; Solution F in Table 1) and after washout (washout). The current was recorded from cell-attached patches. c indicates a closed level. (B) Average NPo of BK channel current. Recordings were made at a holding potential of −10 mV. *Significantly different (P < 0.05) from control values (t-value 25.16). A total of 18 cells isolated from 15 animals were used in the experiment and 14 tests were performed for data collection.

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  Figure 6. Effect of ryanodine and caffeine on BK channel activity. (A) Representative BK channel currents before (control) and after addition of 40 μM ryanodine (ryanodine) to the bath solution. c indicates a closed level. (B) Representative BK channel currents before (control) and after addition of 10 mM caffeine (caffeine) to the bath solution. c indicates a closed level. (C,D) Average NPo of BK channel current. Recordings were made at a holding potential of −10 mV. *Significantly different (P < 0.05) from control values (t-values, ryanodine: 5.91, caffeine: 5.80). A total of 18 cells isolated from a total of 12 animals were used in the experiment, and 12 tests were performed for data collection.

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  Figure 7. Effect of nifedipine and Bay K 8644 on BK channel activity. (A) Representative BK channel currents before (control) and after addition of 5 μM nifedipine (nifedipine) to the bath solution. c indicates a closed level. (B) Representative BK channel currents before (control) and after addition of 5 μM S(-)-Bay K 8644 (BayK8644) to the bath solution. The current was recorded at a Vpip of −10 mV from cell-attached patches. c indicates a closed level. (C,D) Average NPo of BK channel current (t-values, nifedipine: 3.13, BayK8644: 3.13). (E) Representative BK channel currents before (control) and after addition of extracellular 5 μM S(-)-Bay K 8644 (BayK8644) using the backfill method at a Vpip of −60 and 0 mV, respectively, from cell-attached patches. c indicates a closed level. (F) Average NPo of BK channel current. *Significantly different (P < 0.05) from control at +20 mV values.†Significantly different (P < 0.05) from BayK8644 at +20 mV values. A total of 39 cells isolated from a total of 32 animals were used in the experiment, and 25 tests were performed for data collection.