|[Frontiers in Bioscience 1, d214-233, September 1, 1996]|
MOLECULAR BIOLOGY OF THE GABAA RECEPTOR: FUNCTIONAL DOMAINS
IMPLICATED BY MUTATIONAL ANALYSIS
Martin Davies1, Alan N. Bateson1,2 and Susan M. J. Dunn1,2
1 Department of Pharmacology
2Division of Neuroscience,
Faculty of Medicine, University of Alberta, Edmonton, Alberta,
Canada T6G 2H7
2Division of Neuroscience, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Received 07/16/96; Accepted 07/22/96; On-line 09/01/96
The GABAAR, like several other LGICS, can be phosphorylated by various protein kinases (for reviews see 21, 106). Analysis of the primary sequences of the GABAAR ß and gamma subunits reveals that, within the presumed intracellular loop between TM3 and TM4, there are consensus sequences for phophorylation by cyclic AMP-dependent kinase (PKA), calcium-calmodulin-dependent kinase (PKC), and tyrosine-protein kinase (PTK). Biochemical studies have revealed that both the ß and gamma subunits can be phosphorylated by PKA in vitro (107, 108) and that the gamma subunit can also be phosphorylated by PKC (109). The functional consequences of phophorylation appear to vary, however, in a tissue-dependent manner. In general, phosphorylation leads to changes in the amplitude, frequency, and desensitization rates of GABA-gated currents.
By using a combination of phosphopeptide analysis and site directed mutagenesis, the phophorylation sites of the ß and gamma subunits have been definitively identified. Phosphopeptide analysis of the alpha1ß1gamma2 receptor subtype expressed in HEK293 cells demonstrated that phophorylation by PKA occurred exclusively on serine residues of the ß1 subunit (110-112). Mutational analysis of these residues in the intracellular loop of the ß1 subunit identified a serine at position 409 as being the substrate for PKA. Replacement of serine with an alanine resulted in a complete loss of phosphorylation, and prevented modulation of GABA-gated currents by PKA.
A similar approach was taken to identify regions of the ß and gamma subunit that are phosphorylated by PKC (109-112). Mutational analysis showed that PKC phophorylated the same serine in the ß1 subunit intracellular loop as PKA and, as might be expected, produced the same functional consequences. Both gamma2S and gamma2L subunits are phosphorylated by PKC at position 307 in the large intracellular loop. The long and short forms of the gamma2 subunit differ by the absence or presence of a sequence of 8 amino acids within the intracellular loop. Within this sequence in the gamma2L subunit, there is an extra PKC phosphorylation site at position 343. The sensitivity of the GABAAR to ethanol has been shown to be dependent on phophorylation of this residue (113, 114). Phosphorylation of either of the two PKC sites in the gamma2L subunit produced slightly different regulation of GABA-gated current than was seen when only the ß subunit was phosphorylated.
Glycosylation appears to be an important facet of the expression and assembly of LGICs, although, at least for the nAChR, it may not be an absolute requirement for cell surface expression (115, 116). The only mutational analysis of this post-translational modification in the GABAAR introduced glutamine residues in the place of the asparagine residues proposed to form N-linked glycosylation sites in the hydrophilic N-terminal domain of the rat alpha1 subunit (117). The substitution of either or both arginines at position 10 and 110 formed a GABAA receptor with normal pharmacology when coexpressed in Xenopus oocytes with ß1 and gamma2 subunits. The whole cell current produced by these mutant receptors was not significantly different than that seen with wild-type receptors, indicating not only that glycosylation was not important for function, but that lack of glycosylation probably did not impair receptor expression. A different picture emerged when the mutated subunit was expressed with ß1 and gamma2 subunits in HEK293 cells. In this system, expression of either the N10Q or N110Q mutations at 37°C resulted in significantly reduced expression levels as measured by [3H]Ro15-1788 binding. Furthermore, the expression level of the double mutant was such that no measurable binding of [3H]Ro15-1788 was detected. Since the Kd values for these mutants were similar, the reduced level of radioligand binding was due to differences in expression levels. Thus, in the HEK293 expression system, appropriate glycosylation is a major component in subunit assembly and expression. Interestingly, the processing of non-glycosylated subunits appears to be temperature dependent. When mutant receptors were expressed in HEK293 cells at 30°C, some of the expression returned to wild type levels.