N-terminal PDZ-binding domain in Kv1 potassium channels

AbstractWe have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95

AMP-activated protein kinase inhibits K_v1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation

KEY POINTS: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (K(v)) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP‐activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited K(v)1.5 channels in pulmonary arterial myocytes. AMPK activation by 5‐aminoimidazole‐4‐carboxamide riboside, A769662 or C13 attenuated K(v)1.5 currents in pulmonary arterial myocytes, and this effect was non‐additive with respect to K(v)1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human K(v)1.5 channels in cell‐free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing K(v)1.5. These results suggest that AMPK is the primary mediator of reductions in K(v)1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (K(v)) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP‐activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1‐dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited K(v) currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte K(v) currents were also markedly inhibited upon AMPK activation by A769662, 5‐aminoimidazole‐4‐carboxamide riboside and C13 and by intracellular dialysis from a patch‐pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2β2γ1 or α1β1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK‐sensitive K(+) currents, which were also blocked by the selective K(v)1.5 channel inhibitor diphenyl phosphine oxide‐1 but unaffected by the presence of the BK(Ca) channel blocker paxilline. Moreover, recombinant human K(v)1.5 channels were phosphorylated by AMPK in cell‐free assays, and K(+) currents carried by K(v)1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates K(v) channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of K(v)1.5 and/or an associated protein

Localization of Kv1.5 in native and heterologous cell systems

Ion channel synthesis, trafficking and localization within the plasma membrane are highly regulated processes. They involve convergence of signals at the level of mRNA synthesis, chaperone-mediated folding and trafficking, interplay of kinases and phosphatases and assembly of macromolecular complexes fixed in place via anchoring proteins connected to the cytoskeleton. Little is known regarding the mechanisms that ensure the efficient surface membrane expression and localization of the potassium ion channel, Kvl.5, within cardiac myocytes or even heterologous cell systems. The work presented here is part of an ongoing attempt to understand these mechanisms. A common protein-protein binding motif has been studied that binds PDZ proteins found to be important for the localization of many membrane proteins. The PDZ protein, PSD-95 but not SAP97, efficiently binds the C-terminal PDZ binding domain of Kvl.5 in yeast two-hybrid assays, GST pull-down experiments, and in coimmunoprecipitations. Both of these PDZ proteins can also regulate channel expression through the N-terminus of the channel. While PSD-95 and a channel C-terminal truncation mutant were co-immunoprecipitated, no direct interaction was detected with SAP97 despite obvious increases in channel surface expression. In the process of developing efficient detection methods for Kvl.5 in native cardiac tissue, the expression and localization of the channel in mammalian cardiac myocytes was defined to understand potential targeting and retention mechanisms. A detailed characterization of antibodies and expression of Kvl.5 in canine cardiac tissue unambiguously demonstrated a physiological role for the channel expressed in the atria and contributing to the repolarization phase of the atrial action potential. A more detailed examination of channel localization failed to find evidence of targeting to specialized membrane domains such as caveolae and/or lipid rafts. At the resolution of fluorescence microscopy only minor colocalization was found with the caveolar protein, caveolin-3, and the channel was absent from light-buoyant fractions along with raft markers in sucrose gradient fractionations. Overall, the work in this thesis has begun to provide insight into the multiple mechanisms regulating Kv channel surface expression and localization. Clearly, such mechanisms will prove to be of central importance in both the physiological and pharmacological control of channel activity in cardiac tissues.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat