Identification, characterization and regulation of sodium channel phosphatases from rat brain
Abstract
Sodium channels, key molecules in controlling neuronal excitability, are inhibited up to 80% by PKA mediated phosphorylation. Knowledge of the identity and regulation of sodium channel phosphatases is important in understanding the control of neuronal excitability. We find that both phosphatase 2A (PP-2A) and calcineurin can dephosphorylate sodium channels. Calcineurin requires Ca$\sp{2+}$ for activity. Since little is known about PP-2A regulation we explored mechanisms in brain which regulate its sodium channel phosphatase activity. Two PP-2A regulatory mechanisms involve the association of B regulatory subunits and activation by ceramide. PP-2A is a trimeric enzyme containing a catalytic subunit, an A subunit and one of several B subunits. The B subunit can be dissociated by treatment with heparin. We find that heparin stimulates the sodium channel phosphatase activity of trimeric PP-2A but not that of dimeric or monomeric PP-2A. These data demonstrate that the B subunit can alter the sodium channel phosphatase activity of PP-2A. Since the "trimer" used in these experiments was a mixture of heterotrimers, how individual B regulatory subunits alter sodium channel phosphatase activity is unknown. Fractionation of rat brain extracts reveals at least 4 distinct forms of PP-2A differing in the B subunit which they contain. Current studies are directed toward purifying each of these trimeric holoenzymes and evaluating their sodium channel phosphatase activity. Ceramide is a lipid which activates PP-2A. Our studies show that ceramide activates monomeric PP-2A. This observation demonstrates that the catalytic subunit contains the ceramide binding site and raises the possibility that other serine/threonine phosphatases may be regulated by lipid messengers. Importantly, ceramide activates PP-2A toward sodium channels. Further studies should be directed toward determining whether ceramide regulates sodium channel dephosphorylation in neurons.
Degree
Ph.D.
Advisors
Rossie, Purdue University.
Subject Area
Biochemistry|Neurology|Anatomy & physiology|Animals|Cellular biology
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