alpha2-Adrenoceptor-mediated potassium currents in acutely dissociated rat locus coeruleus neurones

Abstract

1. The noradrenaline (NA)-activated response was investigated in neurones acutely dissociated from the rat locus coeruleus (LC) using nystatin-perforated, conventional whole-cell and inside-out patch recording modes under current- and voltage-clamp conditions. 2. Under current-clamp conditions, NA hyperpolarized the LC neurones, abolishing the spontaneous action potentials. In voltage-clamp studies, NA induced an inwardly rectifying K+ current (INA) in a concentration-dependent manner with a half-maximum effective concentration of 2.2 x 10(-7) M. 3. INA was mimicked by the alpha2-agonist UK14304 but was inhibited by either the alpha2B/alpha2C antagonist ARC239 or the alpha1- and alpha2B/alpha2C antagonist prazosin, suggesting the contribution of alpha2B/alpha2C adrenoceptors. 4. INA was inhibited by the intracellular application of GDPbetaS but fully activated by intracellular perfusion of GTPgammaS. 5. In the inside-out recording mode, the application of GTP to the cytoplasmic side of the patch membrane markedly enhanced the open probability of the NA-activated single channels which represented the inwardly rectifying properties. 6. These results indicate that the activation of alpha2B/alpha2C adrenoceptors coupled with GTP-binding protein directly activates the inwardly rectifying K+ currents in rat LC neurones, thus resulting in a decrease in the spontaneous firing activities.

Figure 6. Effects of GDPβS and GTPγS on I_NA

The neurones were intracellularly perfused with an internal solution containing each guanine nucleotide analogue by using the conventional whole-cell patch recording mode. All recordings were performed in external solution containing 20 mm K⁺ at a V_H of −80 mV. A, representative recordings of I_NA after intracellular perfusion with internal solutions containing 0.3 mm GTP (a), 0.3 mm GDPβS (b) or 0.3 mm GTPγS (c). The horizontal bars indicate the period of 3 × 10⁻⁷ m NA application. The numbers above the horizontal bars show the time of intracellular dialysis. B, the relationship between the time of intracellular perfusion with GTP or GDPβS and the relative peak amplitude of I_NA. All responses were normalized to the peak amplitude of I_NA with intracellular perfusion for 3 min. Each point and the vertical bars represent the mean ±s.e.m. from 4 neurones.

Figure 5. Effects of K⁺ channel blockers on I_NA

All recordings were performed in external solution containing 20 mm K⁺ at a V_H of −80 mV. The neurones were pretreated for 1 min with each K⁺ channel blocker before the simultaneous application of 3 × 10⁻⁷ m NA. A, 4-aminopyridine (4-AP) inhibited I_NA in a concentration-dependent manner. B, the concentration-inhibition relationships for various K⁺ channel blockers on I_NA. All responses were normalized to the peak current induced by 3 × 10⁻⁷ m NA alone. Each point and the vertical bars represent the mean ±s.e.m. from 5 neurones.

Figure 1. Effect of NA on the membrane potential of an LC neurone

A, spontaneous action potentials recorded in the perforated patch recording configuration. NA (3 × 10⁻⁸, 3 × 10⁻⁷ and 3 × 10⁻⁶ m) was added to the external solution as indicated by the bars. The action potentials are not fully resolved because of the insufficiently high frequency response of the chart recorder. B, digitized potential traces at symbols a-c indicated in A.

Figure 2. Activation of the inwardly rectifying K⁺ current by NA

A, NA (10⁻⁶ m)-induced currents at various holding potentials (V_H) in the same neurone perfused with external solution containing 5 mm K⁺. B, the current–voltage (I–V) relationship of NA-induced responses in external solution with three different K⁺ concentrations (5, 20 or 50 mm). The intracellular K⁺ concentration ([K⁺]_i) was 120 mm throughout the experiments. All responses were normalized to the peak current induced by NA in external solution containing 20 mm K⁺ at a V_H of −80 mV (*). The I–V curves of the NA-induced responses showed inward rectification. Each point and the vertical bars represent the mean ±s.e.m. from 5 neurones. The inset shows the relationship between the external K⁺ concentration ([K⁺]_o) and the reversal potential (E_NA) of NA-induced current. Each E_NA was estimated based on the I–V relationship.

Figure 3. Inward currents induced by adrenoceptor agonists

All recordings were performed in external solution containing 20 mm K⁺ at a V_H of −80 mV. A, the NA-induced currents at various concentrations. B, the concentration-response relationship for various adrenoceptor agonists. All responses were normalized to the peak current induced by 10⁻⁶ m NA (*). Adrenaline and UK14304 mimicked the NA response. Each point and the vertical bars represent the mean ±s.e.m. from 5 neurones.

Figure 4. Effects of adrenoceptor antagonists on the NA-induced current (I_NA)

All recordings were performed in an external solution containing 20 mm K⁺ at a V_H of −80 mV. The neurones were pretreated for 15 s with each adrenoceptor antagonist before the simultaneous application of 10⁻⁶ m NA. A, yohimbine inhibited I_NA in a concentration-dependent manner. B, the concentration-inhibition relationships for various NA antagonists on I_NA. All responses were normalized to the peak current induced by 10⁻⁶ m NA alone. Each point and the vertical bars represent the mean ±s.e.m. from 5 neurones.

Figure 7. Effect of NEM on I_NA

All recordings were performed in external solution containing 20 mm K⁺ at a V_H of −80 mV. A, I_NA induced by 3 × 10⁻⁷ m NA was suppressed after the perfusion of external solution containing 5 × 10⁻⁶ m N-ethylmaleimide (NEM) for 2 min, whereas the 3 × 10⁻⁵ m glycine-induced response was not. B, the current amplitudes of NA- and glycine-induced responses before and after 2 min of NEM treatment were compared. Each column and the vertical bars represent the mean ±s.e.m. from 7 neurones.

Figure 8. Effect of GDP-GTP exchange on the NA-induced single channel current

The inside-out patch recording mode was used to record the NA-induced single channel current. The patch pipette contained 10⁻⁷ m NA. The membrane potential (V_m) was held to −60 mV. A, 0.1 mm GDP and GTP were added to the bath solution as indicated by the horizontal bar above the record. B, digitized current traces obtained from A. The continuous lines show the closed level (c) and the dashed lines show the open level (o).

Figure 9. The I-V relationship of the NA-induced single channel current

A, the NA-induced single channel current recordings obtained at various V_m values. The patch pipette contained 10⁻⁷ m NA. The continuous lines show the closed level and the dashed lines show the open level. B, I-V relationship of the NA-induced single channel currents. Each point and the vertical bars show the mean ±s.e.m. from 5 neurones. The inset shows an amplitude histogram of the currents recorded at −60 mV. The channel openings were fitted to the Gaussian curve with a peak value of −1.92 pA.