Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels

Abstract

Phosphorylation is the most common and abundant post-translational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies in revealing the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events.

Figure 1. Mechanisms of Kv channel modulation by phosphorylation in neurons

Reversible changes in phosphorylation of dendritic and axonal Kv channels can dynamically modulate their expression, localization and activity different diverse mechanisms, such as a) regulating intracellular trafficking (e.g. Kv1.2, Kv4.2), b) distribution within the plasma membrane (e.g. Kv2.1), and/or c) direct modulation of voltage-dependent gating (e.g. Kv2.1, Kv3.1, Kv7.2, Kv4.2). Kv channels primarily expressed in somatodendritic regions are in red, and those in axons in green.

Figure 2. Kv1.2 and Kv2.1 are highly phosphorylated in rat brain

Phosphorylation in Kv1.2 and Kv2.1 channels causes altered electrophoretic mobility on SDS-PAGE. Phosphorylation is revealed by differences in electrophoretic migration of these proteins in samples of rat brain membranes (RBM) analyzed by immunoblot without (-) or with (+) prior alkaline phosphatase (AP) treatment of the RBM.

Figure 3. Excitatory stimulation causes changes in Kv2.1 localization in rat hippocampal neurons

Glutamate treatment (10 μM, 15 min) of cultured hippocampal neurons induces dispersion of clusters of Kv2.1 (green). Neurons were double stained with anti-MAP2 antibody (red) as a neuronal somatodendritic marker.