Graded contribution of the Gbeta gamma binding domains to GIRK channel activation

Abstract

G protein coupled inwardly rectifying K(+) channels (GIRK/Kir3.x) are mainly activated by a direct interaction with Gbetagamma subunits, released upon the activation of inhibitory neurotransmitter receptors. Although Gbetagamma binding domains on all four subunits have been found, the relative contribution of each of these binding sites to channel gating has not yet been defined. It is also not known whether GIRK channels open once all Gbetagamma sites are occupied, or whether gating is a graded process. We used a tandem tetrameric approach to enable the selective elimination of specific Gbetagamma binding domains in the tetrameric context. Here, we show that tandem tetramers are fully operational. Tetramers with only one wild-type channel subunit showed receptor-independent high constitutive activity. The presence of two or three wild-type subunits reconstituted receptor activation gradually. Furthermore, a tetramer with no GIRK1 Gbetagamma binding domain displayed slower kinetics of activation. The slowdown in activation was found to be independent of regulator of G protein signaling or receptor coupling, but this slowdown could be reversed once only one Gbetagamma binding domain of GIRK1 was added. These results suggest that partial activation can occur under low Gbetagamma occupancy and that full activation can be accomplished by the interaction with three Gbetagamma binding subunits.

Fig 1.

Tet(4-1-4-1) gates similarly to wild-type channels. (A) Wild-type monomers GIRK1 (gray) and GIRK4 (black) were coexpressed in Xenopus oocytes with m2R. (Left) Continuous recording of current at −80 mV. Oocytes were bathed in 90K solution, followed by application of 3 μM carbachol to activate the channels. Carbachol was washed out with 90K, and then GIRK channels were selectively blocked by the application of 1 mM barium. (Right) Single-channel currents from oocytes injected with GIRK1, GIRK4, and the Gβ1γ2 subunits under the cell-attached configuration. The holding potential was −100 mV. Channel openings are shown as downward deflections. (B) Tet(4-1-4-1) is composed of two subunits of GIRK4 alternating with two subunits of GIRK1. (Left) Currents were recorded in the same manner as in A. (Inset) Western blot for Tet(4-1-4-1) expressed in oocyte membranes shows a band at approximately 200 kDa, corresponding to the estimated size the tetramer. (Right) Single-channel currents from oocytes expressing Tet(4-1-4-1) and the Gβ1γ2 subunits. (C) Tet(0-0-0-0) is composed of GIRK1/IRK1 chimeras, where the N- and C-terminal domains of GIRK1 were replaced with the corresponding IRK domains, and GIRK4/IRK1 with similar substitution, to make a nongating tetramer (see text). Continuous recordings and single-channel currents were recorded as described above, although single-channel activity was recorded in the absence of Gβγ.

Fig 2.

A graded response of tetramers upon induction by carbachol. Currents at −80 mV were recorded in 90K solution with and without 3 μM carbachol. The average of the basal currents for each tetramer (three to five batches of oocytes, n > 6 per batch) was arbitrarily set at 100% (white bars), and the amount of agonist-induced increase in current upon the addition of 3 μM carbachol was determined (black bars). (Inset) Tetramers were grouped according to the number of constitutively active subunits they contain; Tet(4-1-4-1) contains none, Tet(4-1-4-0) and Tet(0-1-4-1) contain one, Tet(0-1-4-0), Tet(0-0-4-1), Tet(4-1-0-0), Tet(4-0-4-0), and Tet(0-1-0-1) contain two, Tet(0-1-0-0) and Tet(0-0-4-0) contain three, and Tet(0-0-0-0) contains four. The ability to gate was defined as the fold current increase above basal activity in tetramers containing specific number of constitutively active subunits, divided by the fold-induction of Tet(4-1-4-1) and expressed as a percentage.

Fig 3.

Kinetics of activation of the various tetramers. Half-maximal time for activation (t_1/2) was calculated by determining the time it takes for currents to reach half the maximum of activated currents (difference between induced and basal levels). Holding potential was −80 mV. Light gray bars denote measurements from oocytes that also coexpress RGS4. Data were averaged over one to three batches of oocytes.

Fig 4.

Slow activation of Tet(4-0-4-0) is independent of G protein or receptor type. Tet(4-0-4-0) or Tet(4-1-4-1) were coexpressed in the same oocytes with all three G_i/o-coupled receptors: m2R, mGluR₂, and κ opioid. The agonists used were carbachol, K-glutamate, and , respectively. Separately, Tet(4-0-4-0) or Tet(4-1-4-1) was coexpressed with β₂AR and Gαs, and currents were induced with isoproterenol at a holding potential of −80 mV. t_1/2 was determined as described in the Fig. 3 legend.