Substitution of a cysteine in the extracellular mouth of the pore of the Shaker-delta K+ channel permits allosteric inhibition of the channel by Zn2+ or Cd2+ ions at micromolar concentrations. Cd2+ binds weakly to the open state but drives the channel into the slow (C-type) inactivated state, which has a Kd for Cd2+ of approximately 0.2 microM. There is a 45,000-fold increase in affinity when the channel changes from open to inactivated. These results indicate that C-type inactivation involves a structural change in the external mouth of the pore. This structural change is reflected in the T449C mutant as state-dependent metal affinity, which may result either from a change in proximity of the introduced cysteine residues of the four subunits or from a change of the exposure of this residue on the surface of the protein.

GABA and the GABAB receptor agonist baclofen activated a potassium conductance in acutely dissociated hippocampal CA3 neurons. Baclofen-activated current required internal GTP, was purely potassium selective, and showed strong inward rectification. As with acetylcholine-activated current in atrial myocytes, external Cs+ blocked inward but not outward current in a highly voltage-dependent manner, whereas Ba2+ blocked with no voltage dependence. Unlike the cardiac current, however, the baclofen-activated current showed no intrinsic voltage-dependent relaxation. With fast solution exchange, current was activated by baclofen or GABA with a lag of approximately 50 msec followed by an exponential phase (time constant approximately 225 msec at saturating agonist concentrations); deactivation was preceded by a lag of approximately 150 msec and occurred with a time constant of approximately 1 sec. GABA activated the potassium conductance with a half maximally effective concentration (EC50) of 1.6 microM, much lower than that for activation of GABAA receptor-activated chloride current in the same cells (EC50 approximately 25 microM). At low GABA concentrations, activation of the GABAB current had a Hill coefficient of 1.4-2.1, suggesting cooperativity in the receptor-to-channel pathway. Although the maximal conductance activated by GABAB receptors is much smaller than that activated by GABAA receptors, its higher sensitivity to GABA and slower time course make it well suited to respond to low concentrations of extra-synaptic GABA.

We characterized potassium current activated by G-proteincoupled receptors in acutely dissociated hippocampal CA3 neurons. Agonists for serotonin, adenosine, and somatostatin receptors reliably activated a potassium-selective conductance that was inwardly rectifying and that was blocked by 1 mM external Ba 2ϩ . The conductance had identical properties to that activated by GABA B receptors in the same cells. In onehalf of the CA3 neurons that were tested, the metabotropic glutamate agonist 1S,3R-ACPD also activated inwardly rectifying Ba 2ϩ -sensitive potassium current. Activation of the current by serotonin and adenosine agonists occurred with a time constant of 200-700 msec after a lag of 50-100 msec; on removal of agonist the current deactivated with a time constant of 1-2 sec after a lag of 200-400 msec. These kinetics are similar to GABA B -activated current and consistent with a direct action of G-protein on the channels. For somatostatin, both activation and deactivation were approximately fourfold slower, probably limited by agonist binding and unbinding. The halfmaximally effective agonist concentrations were ϳ75 nM for somatostatin, ϳ100 nM for serotonin, and ϳ400 nM for 2-chloroadenosine. Dose-response relationships had Hill coefficients of 1.2-1.9, suggesting cooperativity in the receptor-tochannel coupling mechanism. At saturating concentrations of agonists, the combined application of baclofen and either somatostatin, serotonin, or 2-chloroadenosine produced effects that were subadditive and often completely occlusive. However, at subsaturating concentrations the effects of baclofen and 2-chloroadenosine were supra-additive. Thus, low levels of different transmitters can act synergistically in activating inwardly rectifying potassium current.