In liver tissue, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of LKB1, MO25α/β, and STRADα/β proteins; however, this complex has yet to be characterized in skeletal muscle. In this report, we demonstrate the expression of the LKB1-MO25-STRAD AMPKK complex in adult skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these complex-forming proteins. To facilitate this characterization we used control (ctrl) and muscle-specific LKB1 knockout (LKB1-/-) mice. LKB1 detection in untreated ctrl and LKB1-/- muscle lysates revealed two protein bands at approximately 50 and 60 kDa; although, only the heavier band was significantly diminished in LKB1-/- samples (ctrl: 55±2.5 AU; LKB1-/-: 13±1.5 AU; p<0.01), suggesting that LKB1 is not represented at 50 kDa as cited previously. Detection of LKB1 at the higher molecular weight was further confirmed following purification of the AMPKK complex using polyethylene glycol (PEG) (ctrl: 43±5 AU; LKB1-/-: 8.4±4 AU; p<0.01). Following ion-exchange-fast protein liquid chromatography (FPLC) the low protein band was undetectable in ctrl and LKB1-/- fractions. Mass spectrometry of PEG-treated ctrl lysates confirmed LKB1 protein detection in the 60 kDa protein band while none was detected in the 50 kDa band. Co-immunoprecipitation assays demonstrated associations between all combinations of LKB1, MO25, and STRAD in LKB1-positive samples, confirming proper complex formation. Quantitative-PCR revealed significantly reduced expression of MO25α and STRADβ in LKB1-/- muscle. Lastly, detection of CaMKKα/β protein in ctrl and LKB1-/- muscle lysates confirmed the presence of another AMPKK in muscle. Interestingly, CaMKKβ protein is increased in LKB1-/- muscle (ctrl: 19±4.3 AU; LKB1-/-: 47±9.2 AU; p<0.05) without an increase in mRNA levels, suggesting compensation for null LKB1 expression. In all, these findings confirm the presence of the LKB1-MO25-STRAD complex in adult skeletal muscle, suggest a novel post-translational modification of LKB1, and identify a potential compensatory mechanism for loss of LKB1 protein in skeletal muscle.

Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.