Evidence for phosphorylation-dependent internalization of recombinant human rho1 GABAC receptors

1. Recombinant wild-type or mutant human rho1 GABA receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS-7 cells and studied using the patch clamp technique. 2. Standard whole-cell recordings with 4 mM Mg-ATP in the patch pipette induced a time-dependent decrease in the GABA-activated current (IGABA) amplitude that was not the result of a decrease in GABA sensitivity. In contrast, IGABA 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 microM) or staurosporine (20 nM) prevented the time-dependent decrease in the amplitude of IGABA 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 IGABA. Although the protein kinase C (PKC) activator 4beta-phorbol 12-myristate, 13-acetate (PMA) did not reduce the amplitude of IGABA, inclusion of the catalytic domain of PKC in the recording pipette accelerated the time-dependent decrease in current amplitude. These data suggest that phosphorylation is involved in the regulation of the amplitude of IGABA. 4. Mutation of the three PKC consensus sequences of the rho1 receptor had no significant effect on the decline in IGABA, indicating that direct phosphorylation of these putative sites on the rho1 receptor does not underlie the time-dependent decrease in amplitude. 5. In COS-7 cells transfected with wild-type rho1 receptors, the amplitude of IGABA had completely recovered to the original value when the same cells were repatched after 30-40 min, indicating that the decline in IGABA was 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 inactivation suggesting that the actin cytoskeleton may play a role in the regulation of the amplitude. 7. Coincident with the decrease in the amplitude of IGABA, the cell capacitance significantly decreased in the presence of ATP in the patch pipette. This decrease in capacitance was not observed in the absence of Mg-ATP. The decrease in the membrane surface area suggests that receptor internalization could be a potential mechanism for the observed inactivation. 8. At 32 C, compared with 22 C, the rate and magnitude of the decline was increased dramatically. In contrast, at 16 C, no significant change in IGABA 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 IGABA. 9. The specificity of the decrease in IGABA was assessed by coexpressing the voltage-dependent potassium channel Kv1.4 along with the rho1 receptor in HEK293 cells. The amplitude of the potassium current (IKv1.4) exhibited very little decrement in comparison to IGABA suggesting that the putative GABA receptor internalization was not the consequence of a non-specific membrane retrieval.