IDENTIFICATION OF REGIONS THAT REGULATE THE EXPRESSION AND ACTIVITY OF G-PROTEIN-GATED INWARD RECTIFIER K+ CHANNELS IN XENOPUS OOCYTES

1. The involvement of the cytoplasmic and core regions of K+ channel K ir3.1 and Kir3.2 subunits in determining the cell surface expression a nd G protein-gated activity of homomeric and heteromeric channel compl exes was investigated by heterologous expression of chimeric and wild- type subunits together with the m(2) muscarinic receptor in Xenopus oo cytes. 2. Go-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression o f these subunits. Immunofluorescence labelling indicated that Kir3.2 h omomeric channels and Kir3.1-Kir3.2 heteromeric channels were expresse d at high levels at the cell surface whereas Kir3.1 homomeric complexe s were not expressed at the cell surface. Chimeric subunits composed o f Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expressio n of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chime ric subunit (121) which displayed dramatically increased acetylcholine -induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chime ra 121 channel openings mere longer than Kir3.2 openings. 4. Individua lly substituting the N- and C-terminal tails of Kir3.1 for those of Ki r3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity o f heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher rat io of ACh-induced to basal activity than the Kir3.1.-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be ac tivated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substitutin g the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core a nd G-terminal regions of Kir3.1 independently conferred time dependenc e on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.