Kobayashi T , Washiyama K , Ikeda K .

	Figure 1. Effects of pregnenolone sulfate (PREGS) on Kir2.3 channels expressed in Xenopus oocytes.(A) Upper row, in an oocyte injected with Kir2.3 mRNA, current responses to 30 µM PREGS and to 50 µM PREGS in the presence of 3 mM Ba2+ are shown. Lower row, in an uninjected oocyte, no significant current responses to 300 µM PREGS and 3 mM Ba2+ are shown. Current responses were measured at a membrane potential of −70 mV in an hK solution containing 96 mM K+. Asterisks show the zero current level. Horizontal bars show the duration of application. (B) Effects of various neurosteroids: PREG, PREGS, DHEA, DHEAS, progesterone (PROG), 17β-estradiol (E2), corticosterone (CORT), 3α-OH-DHP and THDOC, on Kir2.3 channels. The magnitudes of the effect of 100 µM neurosteroids on Kir2.3 channels were normalized to the 3 mM Ba2+-sensitive current components in oocytes expressing Kir2.3 channels (n≥4 for each steroid). Data are expressed as mean±SEM.
	Figure 2. Characteristics of Kir2.3 channel potentiation by PREGS.(A) Concentration-dependent effect of PREGS on Kir2.3 channels. The magnitudes of Kir2.3 currents potentiated by PREGS were normalized to the 3 mM Ba2+-sensitive current components in Xenopus oocytes expressing Kir2.3 channels (554.0±79.9 nA, n = 6). Data are expressed as mean±SEM of the percentage responses. (B) Correlation between amplitudes of current response to 30 µM PREGS and amplitudes of the 3 mM Ba2+-sensitive current components in oocytes expressing Kir2.3 channels. The correlation coefficient was 0.894 (P<0.05, n = 22, regression analysis). Current responses were measured at a membrane potential of −70 mV in an hK solution containing 96 mM K+. (C) Representative Kir2.3 currents elicited by a voltage step to −100 mV for 2 s from a holding potential of 0 mV in the presence or absence of 30 µM PREGS in an oocyte injected with Kir2.3 mRNA. Arrow indicates the zero current level. (D) Current-voltage relationships of 3 mM Ba2+-sensitive currents and 30 µM PREGS-enhanced currents in oocytes expressing Kir2.3 channels. Current responses were normalized to the 3 mM Ba2+-sensitive current component measured at a membrane potential of −100 mV (1510.0±209.9 nA, n = 6).
	Figure 3. Effect of intracellular PREGS in Xenopus oocytes expressing Kir2.3 channels.(A) Comparison of basal Kir2.3 currents before and after PREGS injection in oocytes expressing Kir2.3 channels. The amplitude of Kir2.3 currents was normalized to the amplitude of 3 mM Ba2+-sensitive current components before PREGS injection. (B) Comparison of 50 µM PREGS-induced Kir2.3 currents before and after PREGS injection. Data are expressed as mean±SEM.
	Figure 4. Effects of PREG and DHEAS on PREGS-induced Kir2.3 currents.(A) Representative current responses to 30 µM PREGS and to 30 µM PREGS in the presence of 100 µM PREG in a Xenopus oocyte expressing Kir2.3 channels. Current responses were measured at a membrane potential of −70 mV in an hK solution containing 96 mM K+. (B) Comparison of PREGS-induced Kir2.3 currents in the presence or absence of PREG or DHEAS. Concentrations of PREGS, PREG, and DHEAS were 30, 100, and 100 µM, respectively. Current responses to PREGS in the presence of PREG or DHEAS were normalized to the amplitude of PREGS-induced currents in the absence of PREG or DHEAS (control). Data are expressed as mean±SEM.
	Figure 5. Concentration-response relationships for potentiation of Kir2.3 channels by PREGS at different pH values.The magnitudes of potentiation of Kir2.3 currents by PREGS in oocytes expressing Kir2.3 channels were normalized to the 3 mM Ba2+-sensitive current components, which were 426.9.6±41.4 nA (pH 6.0), 554.0±79.9 nA (pH 7.4) and 729.2±36.6 nA (pH 9.0). The EC50 and nH values were 16.1±1.2 µM and 1.44±0.07 (pH 6.0, n = 10), 15.6±0.9 µM and 1.43±0.03 (pH 7.4, n = 6), and 17.1±1.5 µM and 0.70±0.03 (pH 9.0, n = 8), respectively. Current responses were measured at a membrane potential of −70 mV in an hK solution containing 96 mM K+. Data are expressed as mean±SEM of the percentage responses.
	Figure 6. Comparison of the effects of PREGS on Kir1.1, Kir2.1, Kir2.2, Kir2.3, and Kir3 channels expressed in Xenopus oocytes.The magnitudes of change in Kir currents by 100 µM PREGS were normalized to the 3 mM Ba2+-sensitive current components. For Kir3 channels, oocytes expressing brain-type Kir3.1/Kir3.2 channels were used. Current responses were measured at a membrane potential of −70 mV in an hK solution containing 96 mM K+. Data are expressed as mean±SEM.
	Figure 7. Comparison of the potentiation effects of PREGS on Kir2.3, Kir2.1/Kir2.3, and Kir2.2/Kir2.3 channels expressed in Xenopus oocytes.Responses to PREGS at different concentrations were normalized to the maximal response to PREGS. Current responses were measured at a membrane potential of −70 mV. Data are expressed as mean±SEM.

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