Abstracts

Rationale: Emerging evidence indicates that loss-of-function mutations in the protein KCC2 can cause epilepsy during infancy with migrating focal seizures (EIMFS) (1). KCC2 is the neuron-specific K+/Cl– cotransporter, and it comprises the major mechanism by which neurons extrude Cl– to setup hyperpolarizing GABAA currents. GABAA receptors are the primary mediators of fast inhibitory synaptic transmission and the targets for numerous therapeutic agents including barbiturates, benzodiazepines, and the anesthetic propofol (2). Thus, these mutations will have subsequent detrimental impacts on inhibitory synaptic signaling and the efficacy of widely used drugs, which has further motivated researchers to discover potentiators of KCC2 function. Currently, the only known mechanism to potentiate KCC2 function is by altering its phosphorylation state. The WNK-SPAK kinase pathway directly phosphorylates and inhibits KCC2, and thus inhibiting this pathway can increase KCC2 function (disinhibition), but it is unknown if any these mutations exhibit normal phosphorylation states or if they can be potentiated by kinase inhibitors. Methods: Here we will examine several human KCC2 mutations including the L288H, W318S, S748 deletion, and the E50-Q93 deletion mutant. We will examine how these mutations affect the various biochemical and electrical properties of KCC2 in cell lines and neurons. Specifically, we will examine the expression levels, phosphorylation state, and cell surface levels, as well as changes in GABAA (EGABA) and Glycine (EGLY) receptor reversal potentials. In addition, we will use the selective KCC2 inhibitor VU0463271 to estimate the level of Cl– extrusion, and the WNK kinase inhibitor WNK463 to determine if each mutant can respond to phospho-regulation.   Results: Our experiments in neurons indicated that all the mutations tested thus far cause a reduced amount of total KCC2 protein (relative to WT: W318S 66  8%, p=0.0044, n=3; S748del 5  1%, p<0.0001, n=3), with the S748del mutation exhibiting a more severe reduction compared to W318S (p=0.0002). Cell surface levels of the mutants were also all reduced compared to WT controls (W318S 43  5%, p<0.0001, n=3; S748del 12  2%, p<0.0001, n=3), with the S748del mutation again showing a more severe reduction relative to W318S (p=0.0007). Our functional analysis indicated that under basal conditions only the S748del (EGABA –73  11 mV, n=8 cells) exhibited an impairment compared to WT (EGABA –97  4 mV, n=6 cells, p=0.0293). Preliminary analysis of function in HEK293 cells indicated WNK463 potentiated KCC2 WT activity (EGABA shift –20  7 mV, n=8 cells, p=0.0180, paired t-test). This potentiation also occurred in a self-limiting manner that was highly correlated (r2 = 0.951); cells that exhibited the lowest amount of KCC2 activity at baseline had the highest potentiation by WNK463. Conclusions: Our preliminary data suggest that not all KCC2 mutations cause similar deficits in KCC2 functional expression. Our data could suggest that indirect modulation of KCC2 function could be a viable therapeutic strategy for some but not all EIMFS patients who harbor KCC2 mutations.   Funding: NS101888