Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes

Abstract

The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined. Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21 - 23 degrees C. When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance. NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form. In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential. Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range -80 mV to +20 mV. The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials. When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials. A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face. Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed.

Figure 1

Reversible effect of 7.5 μM NBT on the gating of a single BK channel studied in an inside-out patch of CVSMC membrane. This patch was voltage clamped at a membrane potential of V=+60 mV with [Ca²⁺]_i=1 μM. NBT was applied by perfusion to the cytoplasmic membrane face. 0 and 1 denoted channel closed and channel open states, respectively. Bandwidth of recordings dc-1 kHz.

Figure 2

Effect of NBT on the open time distribution of a single BK channel in an inside-out membrane patch, voltage clamped to V=−20 mV with [Ca²⁺]_i=1 μM. These distributions were plotted as the square root of the number of observations (ordinates) against the logarithm of the open time (abscissae). (A) Open time distribution obtained in Control medium. This distribution contained 396 channel openings and was well described by the sum of two exponential terms (smooth curve) using the following fit parameters, defined in the text. τ_of=0.59 ms; τ_os=7.8 ms, A_of/(A_of+A_os)=0.288. This yielded a mean open time, T_o=5.7 ms. (B) Open time distribution after application of 7.5 μM NBT to the cytoplasmic membrane face. This distribution comprised 840 channel openings. It was well described by a single exponential term (smooth curve) with a time constant, equivalent to T_o, of 0.99 ms.

Figure 3

Influence of NBT on the voltage-dependence of mean channel open time, T_o. The graph shows the relationship between T_o and membrane potential, V in Control medium and in the presence of 7.5 μM NBT, applied to the cytoplasmic face of inside-out membrane patches. Data represent means±s.e.mean from five patches. Asterisks indicate membrane voltages at which mean values of T_o were significantly different in the absence and presence of NBT (P<0.05, ANOVA). [Ca²⁺]_i=1 μM.

Figure 4

The influence of NBT on the relationship between membrane voltage and the mean closed time, T_c of BK channels. NBT (7.5 μM) was applied to the cytoplasmic face of inside-out membrane patches. Data represent means±s.e.mean from eight patches. Asterisks indicate membrane voltages at which mean values of T_c were significantly different in the absence (Control) and presence of NBT (P<0.05, ANOVA). [Ca²⁺]_i=1 μM.

Figure 5

The effect of NBT on the relationship between membrane potential and open probability, P_o of BK channels. NBT (7.5 μM) was applied to the cytoplasmic face of inside-out membrane patches. Data represent means±s.e.mean from eight patches. Asterisks indicate membrane voltages at which mean values of P_o were significantly different in the absence (Control) and presence of NBT (P<0.05, ANOVA). [Ca²⁺]_i=1 μM.

Figure 6

The influence externally applied NBT on the relationship between membrane voltage and the mean open time, T_o of BK channels. NBT (7.5 μM) was applied to the external face of inside-out membrane patches. Data represent means±s.e.mean from eight patches. Asterisks indicate membrane voltages at which mean values of T_o were significantly different in the absence (Control) and presence of NBT (P<0.05, ANOVA). [Ca²⁺]_i=1 μM.

Figure 7

The effect of externally applied NBT on the relationship between membrane voltage and the open probability, P_o of BK channels. NBT (7.5 μM) was applied to the external face of inside-out membrane patches. Data represent means±s.e.mean from eight patches. Asterisks indicate membrane voltages at which mean values of P_o were significantly different in the absence (Control) and presence of NBT (P<0.05, ANOVA). [Ca²⁺]_i=1 μM.