Intermediate conductance Ca2+-activated potassium channels are activated by functional coupling with stretch-activated nonselective cation channels in cricket myocytes

Figures

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  Figure 1. Ba2+-insensitive intermediate conductance single-K+ channel currents in isolated cricket oviduct cells. (A) Two types of single K+ channel current recordings from cell-attached patches under extracellular K+ concentration of 140 mM are shown. Representative single-channel current traces of large conductance Ca2+-activated potassium (BK) channels (left column) and intermediate conductance Ca2+-activated K+ channel (IK) channels are shown when held at +40 mV. Notes at the BK channel currents are sensitive to extracellular (inside the pipette) 1 mM Ba2+ (1Ba). c indicates closed level. (B) Representative single-channel current traces at various membrane potentials (Vm) are shown in the figure. Current activity does not exhibit voltage sensitivity. c indicates closed level. (C) Average single-channel current (i)-Vm relationship (n = 6–12). Data points for Vm from −60 mV to +60 mV were fitted by linear regression, yielding slope conductance values of 59.7 and 61.5 pS for the control and 1Ba, respectively. Cells isolated from a total of 153 animals were used in the experiment and 259 tests were performed for data collection.

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  Figure 2. Single K+ channel I-V relationship at various extracellular K+ concentrations. (A) Mean linear current-voltage (I-V) data at various extracellular K+ concentrations ([K+]o in mM): ●, 140; ▲, 70; ○, 35, from cell-attached patches. (B) Data show a semi-logarithmic plot of [K+]o against the difference between the reversal potential (⊿Erev) obtained at 140 mM and each K+ concentration. Reversal potential (Erev) was obtained by fitting the i-Vm relationship for each K+ concentration in A by linear regression. The slope of the regression line was 58.37 mV/decade. Cells isolated from a total of 53 animals were used in the experiment and 121 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 Vm = 60 mV from excised inside-out patches. c and o indicate closed and open levels, respectively. (B) Average mean open probability (NPO)-[Ca2+]i relationship of steady-state single-channel current. The data fit the Hill equation with EC50 of 88.5 ± 7.8 μM and Hill coefficient of 0.9. Cells isolated from a total of 14 animals were used in the experiment and 41 tests were performed for data collection.

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  Figure 4. Effect of Ca2+ removal from the bath solution and Gd3+ application to the bath solution on IK channel activity. (A, B) (left) Representative IKchannel currents before (control) and after Ca2+ removal from the bath solution (Ca2+-free) or after 30 mM Gd3+ application to the bathsolution. The current was recorded from cell-attached patches at −140 mV. (A, B) (right) Average NPO of IK channel current. *Significantly different (P < 0.05) from control values. Cells isolated from a total of 15 animals were used in the experiment and 25 tests were performed fordata collection.

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  Figure 5. Effect of membrane stretch on IK channel activity. (A, B) Representative stretch-activated (SA) and IK channel currents were recorded at −140 mV from cell-attached mode (n = 12). Recordings were obtained after changing to a Ca2+-free bath solution. The pipette contains Ca2+ in A [Ca2+(+)] but does not contain Ca2+ in B [Ca2+(−)]. c indicates closed level. 1–4 show enlargements in lower and upper traces. Cells isolated from a total of 23 animals were used in the experiment and 43 tests were performed for data collection.

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  Figure 6. Activity relationship between SA and IK due to membrane stretch. (A) Scatter plot of NPO (SA channel activity) was recorded at −140 mV versus membrane suction (n = 48). (B) Scatter plot of NPO (IK channel activity) recorded at −140 mV versus membrane suction (n = 40). (A, B) The values for each relationship were obtained by fitting them with linear regression. The slopes obtained from plots A and B revealed 0.026 and 0.019 cm H2O, respectively. Relationship between NPO and suction, and analyses of the correlation function revealed values of 0.84 and 0.87 from the values of A and B, respectively, indicating a strong positive correlation. (C) Scatterplots of mean NPO versus membrane suction (n = 7) for SA and IK, recorded in A and B, are shown. The correlation coefficient between SA and IK was 0.99. Cells isolated from a total of 83 animals were used in the experiment and 108 tests were performed for data collection.