A G Protein Mediates the Inhibition of the Voltage‐Dependent Potassium M Current by Muscarine, LHRH, Substance P and UTP in Bullfrog Sympathetic Neurons

Abstract

The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extracellular ligands in bullfrog sympathetic neurons was examined using the hydrolysis resistant nucleotide analogues GTPgammaS and GDPbetaS. Im was recorded in large (40 - 60 microm) isolated neurons using the patch-clamp technique in the whole-cell configuration, as well as in neurons from the intact ganglion impaled with conventional microelectrodes. In whole-cell recordings Im could be recorded without significant loss for 1 h or more provided ATP was present in the patch pipette. Muscarine, D-Ala6-LHRH, substance P and UTP reversibly inhibited Im in isolated control neurons, with full and rapid recovery of the current following agonist washout. Dialysis of isolated neurons with various concentrations of GTPgammaS (1 - 100 microM) affected, in a dose-dependent manner, the recovery of Im after its inhibition by brief agonist application. With 50 microM GTPgammaS, Im inhibition became completely irreversible. Similarly, the reversibility of Im inhibition by muscarine was reduced or abolished by the iontophoretic injection of GTPgammaS through a second microelectrode into neurons of the intact ganglion. GTPgammaS by itself caused a slow, agonist-independent suppression of Im in dialysed neurons, thus mimicking agonist action. Dialysis of isolated neurons with GDPbetaS (100 - 500 microM) attenuated by half or more the magnitude of Im inhibition by agonist as compared to control neurons. In addition, GDPbetaS attenuated the response of a given neuron to muscarine and D-Ala6-LHRH, and caused slow increase of Im, as a function of dialysis time. Incubation (2 - 72 h, 4 - 36 degrees C) of isolated neurons or intact ganglions with activated pertussis toxin had no effect on the response to muscarine. Toxin injections to experimental animals were equally ineffective. In contrast to Im, the additional inward current with increase in conductance induced by muscarine and D-Ala6-LHRH reversed with agonist washout in GTPgammaS-dialysed neurons, although more slowly than in control neurons. The results in this study indicate that a G protein, possibly pertussis toxin-insensitive, provides a common coupling step linking muscarinic, substance P, D-Ala6-LHRH and UTP receptors to the inhibition of M current.

On the role of arachidonic acid in M-current modulation by muscarine in bullfrog sympathetic neurons

“…8A). The product p; i is smaller for channels in the Tacit pool than for channels in the Fluent pool, and is zero for channels in the Inhibited pool because the current can be totally suppressed in cells dialyzed with GTPyS (Pfaffinger, 1988;Brown et al, 1989;Lopez and Adams, 1989).…”

“…AA increased Z, after treatment with 4-BPB, indicating that 4-BPB was probably preventing AA release. At low concentrations, NDGA is a relatively specific lipoxygenase inhibitor (but see Vacher et al, 1989;Kom and Horn, 1990), suggesting that a lipoxygenase metabolite participates in the termination of inhibition, possibly by altering the function of the G-protein (Scherer and Breitwieser, 1990) involved in inhibition (Pfaffinger, 1988;Lopez and Adams, 1989;Tokimasa and Akasu, 1990). Taken together, these results suggest that, as found in Aplysia neurons (Carlson and Levitan, 1990), there is a rapid turnover of AA, creating steady state conditions with relatively low concentrations of AA (so IA can be observed) and relatively high concentrations of a lipoxygenase metabolite, such that under resting conditions the rate of termination of inhibition is fast.…”

“…Both cases can be distinguished kinetically. In the first case the rate of recovery will be changed slightly, while in the latter the recovery rate will become very slow [an extreme case is the occlusion of overrecovery when Z, is inhibited irreversibly by muscarine in cells dialyzed with GTPyS (e.g., Pfaffinger, 1988;Lopez and Adams, 1989)]. The effects on the time course of modulation of several pharmacological agents known to interfere with the AA cascade were examined.…”

Inositol Triphosphate-Mediated Ca2+Signals Direct Purinergic P2Y Receptor Regulation of Neuronal Ion Channels

“…Along with muscarine and several peptides, purinergic agonists were soon identified as regulators of neuronal excitability (Siggins et al, 1977) via depression of M current (Adams et al, 1982;Lopez and Adams, 1989;Tokimasa and Akasu, 1990) in frog sympathetic neurons. The mechanism of action of neuronal P2Y receptors on I M has been controversial and may depend on precise neuronal type or receptor isoform.…”

Streptozotocin, an inducer of NAD+ decrease, attenuates M‐potassium current inhibition by ATP, bradykinin, angiotensin II, endothelin 1 and acetylcholine in NG108‐15 cells

“…The M-current inhibition is elicited by a number of different transmitters, including bradykinin [10], ATE UTP [7], angiotensin II [19], endothelin 1 [15], substance P and LHRH [13]. However, the signal transduction pathway from these receptors to M channels is not yet clear [5,18].…”