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Keywords:

  • whole-cell;
  • voltage-clamp;
  • neurotransmitter;
  • modulation

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 GTPγS and GDPβS. Im was recorded in large (40–60 μm) 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 GTPγS (1–100 μM) affected, in a dose-dependent manner, the recovery of Im after its inhibition by brief agonist application. With 50 μM GTPμS, Im inhibition became completely irreversible. Similarly, the reversibility of Im inhibition by muscarine was reduced or abolished by the iontophoretic injection of GTPμS through a second microelectrode into neurons of the intact ganglion. GTPμS by itself caused a slow, agonist-independent suppression of Im in dialysed neurons, thus mimicking agonist action. Dialysis of isolated neurons with GDPβS (100—500 μM) attenuated by half or more the magnitude of Im inhibition by agonist as compared to control neurons. In addition, GDPβS 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°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 GTPγS-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.