adenyl nucleotides. Structural support for this emerged from the
mapping of pathogenicγ2-mutations onto equivalent residues in a
mammalianγ1-containing core AMPK complex. This revealed that
almost all the mutations examined involved amino acids with side
chains close to the adenyl binding site, the majority in direct
contact with AMP/ATP phosphate groups [50].
Early biochemical studies explored the effect of such CBS
domain mutations on AMPK activity using a variety of experimen-
tal approaches, including transient or stable expression of aγ1-
mutation (Arg70Gln, equivalent to Arg302Gln in γ2or
Arg200Gln inγ3) in COS cells or pulmonary fibroblasts, respec-
tively [51]; introduction of the Thr400Asn and Asn488Ile γ2-
mutations into the yeastγ-subunit homologue (Snf4) with analysis
of interaction with Snf1 (α-subunit homologue) [13]; and transient
transfection of WT or mutantγ3 (Arg225Gln) into COS cells
[52]. These reports suggested the primary consequence ofγ2-
mutations to be that of enhanced basal activation of AMPK com-
plexes. Other studies transfecting CCL13 cells with a series of
mutantγ2-variants noted defective activation of AMPK by AMP
in vitro but without evidence for a significant effect on holoenzyme
activity under basal conditions [18, 53]. A subsequent report char-
acterizing the biochemical impact of an Arg531Gln missense muta-
tion inγ2—identified as a sporadic mutation in three neonates with
a fulminant and ultimately fatal clinical course—noted a profound
(>100-fold) reduction in mutant AMPK binding affinity for both
AMP and ATP [26]. When expressed in HEK-293 cells, the
Arg531Gln mutation (together with the identically situated but
biochemically and clinically less severe Arg531Gly mutation) was
found to enhance basal AMPK activation. Critically and in contrast
to studies employing CCL13 cells, this latter study employed a cell
line expressing the major AMPK upstream kinase, LKB1
[26]. Accordingly, these data suggested that while associated with
a reduction or complete loss of sensitivity to AMP and ATP (whose
degree is dependent upon the individual mutation), in the basal
state and in the presence of adequate upstream LKB1,γ2-muta-
tions act to enhance AMPK activity. Notably this mechanism
more readily reconciles with the observed dominant inheritance
pattern, unlike that of a purported loss of function of one allele
of a minorityγ-isoform in the setting of an intact WT allele (i.e.,
haploinsufficiency).4.2 Insights from In
Vivo Models
In parallel with these studies, generation of transgenic mice bearing
humanPRKAG2mutations enabled AMPK activity to be directly
assayed from cardiac tissue extracts. Initially, these also revealed
apparently discrepant findings, with reports of elevatedα1- and
α2-AMPK-associated basal activity in TGN488Imice [32], contrast-
ing with descriptions of reduced total andγ2-specific AMPK activ-
ity in TGR302Q[33] and TGR531G[34] models (the latter with no
difference at age 1 week) (Table2).PRKAG2 syndrome 597