Evidence for phosphorylation-dependent internalization of recombinant human p1 GABA(C) receptors

1. Recombinant wild-type or mutant human rho 1 GABA receptors were expresse d in human embryonic kidney (HEK) 293 or monkey COS-7 cells and studied usi ng the patch clamp technique. 2. Standard whole-cell recordings with 4 mM Mg-ATP in the patch pipette ind uced a time-dependent decrease in the GABA-activated current (I-GABA) ampli tude that was not the result of a decrease in GABA sensitivity. In contrast , I-GABA remained stable when recordings were obtained using the perforated patch configuration or with standard whole-cell recording and no Mg-ATP in the patch pipette. 3. The inhibitors of serine/threonine protein kinases KN-62 (20 mu M) or st aurosporine (20 nM) prevented the time-dependent decrease in the amplitude of I-GABA seen in the presence of ATP. Alkaline phosphatase (220 U ml(-1)), when added to the patch pipette in the absence of ATP, induced a transient potentiation of I-GABA. Although the protein kinase C (PKC) activator 4 be ta-phorbol 12-myristate, 13-acetate (PMA) did not reduce the amplitude of I -GABA, inclusion of the catalytic domain of PKC in the recording pipette ac celerated the time-dependent decrease in current amplitude. These data sugg est that phosphorylation is involved in the regulation of the amplitude of I-GABA. 4. Mutation of the three PKC consensus sequences of the rho 1 receptor had no significant effect on the decline in I-GABA, indicating that direct phos phorylation of these putative sites on the pi receptor does not underlie th e time-dependent decrease in amplitude. 5. In COS-7 cells transfected with wild-type rho 1 receptors, the amplitude of I-GABA had completely recovered to the original value when the same cel ls were repatched after 30-40 min, indicating that the decline:in I-GABA wa s a reversible process. 6. The inhibitor of actin filament formation cytochalasin B, when added to the patch pipette in the absence of ATP, induced a time-dependent inactivat ion suggesting that the actin cytoskeleton may play a role in the regulatio n of the amplitude. 7. Coincident with the decrease in the amplitude of I-GABA the cell capacit ance significantly decreased in the presence of ATP in the patch pipette. T his decrease in capacitance was not observed in the absence of Mg-ATP. The decrease in the membrane surface area, suggests that receptor internalizati on could be a potential mechanism for the observed inactivation. 8. At 32 degrees C, compared with 22 degrees C, the rate and magnitude of t he decline was increased dramatically. In contrast, at 16 degrees C, no sig nificant change in I-GABA was observed over the 20 min recording time. This marked temperature sensitivity is consistent with receptor internalization as a mechanism for the time-dependent decline in I-GABA. 9. The specificity of the decrease in I-GABA was assessed by coexpressing t he voltage-dependent potassium channel Kv1.4 along with the rho 1 receptor in HEK293 cells. The amplitude of the potassium current (I-Kv1.4) exhibited very little decrement in comparison to I-GABA suggesting that the putative GABA receptor internalization was not the consequence of a non-specific me mbrane retrieval.