Cell Surface Stability of γ-Aminobutyric Acid Type A Receptors: DEPENDENCE ON PROTEIN KINASE C ACTIVITY AND SUBUNIT COMPOSITION

Christopher N Connolly,Josef T Kittler,Philip Thomas,Julia M Uren,Nicholas J Brandon,Trevor G Smart,Stephen J Moss

doi:10.1074/jbc.274.51.36565

Abstract

Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.

Highlights

Type A ␥-aminobutyric acid receptors (GABAA), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of ␣, ␤, and ␥ subunits
To explore these issues further, we have examined the surface stability of ␣1␤2 and ␣1␤2␥2 GABAA receptors modified with an N-terminal reporter epitope and expressed in human embryonic kidney cells (A293)
To examine the cell surface stability of GABAA receptors, we have expressed epitope-tagged receptors in A293 cells, focusing on the cell surface expression of receptors composed of ␣1␤2 and ␣1␤2␥2. We reveal that these two types of GABAA receptors are differentially modulated by protein kinase C (PKC) activity, with ␥2-containing receptors being selectively down-regulated from the cell surface

Summary

DEPENDENCE ON PROTEIN KINASE C ACTIVITY AND SUBUNIT COMPOSITION*

(Received for publication, August 9, 1999, and in revised form, September 28, 1999). Christopher N. GABAA receptors have been associated with clathrin-coated vesicles from brain [9] and downregulate their function in an agonist-dependent (9 –11) and kinase-dependent [12, 13] manner To explore these issues further, we have examined the surface stability of ␣1␤2 and ␣1␤2␥2 GABAA receptors modified with an N-terminal reporter epitope and expressed in human embryonic kidney cells (A293). Native GABAA receptors in cultured hippocampal and cortical neurons were seen to constitutively endocytose from the cell soma Together, these results suggest that the trafficking itinerary of the GABAA receptors may be modulated by a protein kinase C (PKC)-dependent mechanism to differentially regulate surface receptor number

EXPERIMENTAL PROCEDURES

Cell Surface Stability of GABAA Receptors

The affinity of the iodinated antibody for

RESULTS

DISCUSSION