Potassium Current Development and its Linkage to Membrane Expansion During Growth of Cultured Embryonic Mouse Hippocampal Neurons: Sensitivity to Inhibitors of Phosphatidylinositol 3-Kinase and Other Protein Kinases

Abstract

Hippocampal pyramidal neurons express three major voltage-dependent potassium currents, IA, ID, and IK. During hippocampal development, IA, the rapidly activating and inactivating transient potassium current, is detected soon after pyramidal neurons can be morphologically identified. Appearance of IA in developing pyramidal neurons is dependent on contact with cocultured astroglial cells; cultured pyramidal neurons not in contact with astroglial cells have reduced membrane area and IA (Wu and Barish, 1994). We have examined intracellular signaling pathways that could contribute to the regulation of IA development by probing developing pyramidal neurons with kinase inhibitors. We observed that exposure to LY294002 or wortmannin, inhibitors of phosphatidylinositol (PI) 3-kinase, reduced somatic cross-sectional area, neurite outgrowth, whole-cell capacitance, IA amplitude and density (amplitude normalized to membrane area), and immunoreactivity for Kv4.2 and/or Kv4.3 (potassium channel subunits likely to be present in the channels carrying IA). In contrast, exposure to ML-9 or KN-62, inhibitors of myosin light chain kinase or Ca2+-calmodulin-dependent protein kinase II (CaMKII), reduced membrane area and IA amplitude but did not affect IA density or Kv4. 2/3 immunoreactivity to the same extent as inhibitors of PI 3-kinase. Unexpectedly, exposure to bisindolymaleimide I or calphostin C, inhibitors of protein kinase C (PKC), did not affect membrane area or potassium current development. Our data suggest that PI 3-kinases regulate both A-type potassium channel synthesis and plasmalemmal insertion of vesicles bearing these potassium channels. CaMKII appears to regulate fusion of channel-bearing vesicles with the plasmalemma and myosin light chain kinase to regulate centripetal transport of channel-bearing vesicles from the Golgi. We further suggest that astroglial cells exert their influence on pyramidal neuron development through activation of PI 3-kinases.

Calcium–Calmodulin-Dependent Kinase II Modulates Kv4.2 Channel Expression and Upregulates Neuronal A-Type Potassium Currents

“…vesicles with the plasmalemma (Wu et al, 1998). Clearly, from these studies and our own, CaMKII activity is required for maintaining normal cellular excitability in many systems.…”

Glial Cell Line-Derived Neurotrophic Factor and Target-Dependent Regulation of Large-Conductance KCaChannels in Developing Chick Lumbar Motoneurons

“…Multiple signaling pathways typically mediate the actions of neurotrophic factors (Segal and Greenberg, 1996;Soler et al, 1998Soler et al, , 1999Wu et al, 1998;Dolcet et al, 1999;Dryer, 2000, 2002). In LMNs, the phosphatidylinositol (PI) 3-kinase/Akt pathway contributes to the anti-apoptotic effects of GDNF.…”

“…4). These compounds have been shown to be effective in perturbing the normal pattern of cell survival and differentiation in developing neurons Wu et al, 1998;Lhuillier and Dryer, 2000;Encinas et al, 2001). In these experiments, GDNF-treated E8 motoneurons were maintained for 24 hr in the presence or absence of specific protein kinase inhibitors.…”

“…4 A). Finally, because a calmodulin (CaM)-dependent kinase pathway is involved in the survival of LMNs and the functional expression of potassium channels in developing hippocampal neurons Wu et al, 1998), we tested whether similar pathways contribute to GDNFinduced expression of K C a channels. However, KN93, a specific inhibitor of CaM-dependent kinase II, and KN92, an inactive enantiomer, had no effect on GDNF stimulation of K C a channel expression (Fig.…”

“…Again, the difference in the intercellular cascades required for stimulation of macroscopic K C a channels may be related to the fact that different channel complexes are expressed in different cell types. A role for PI3 kinase in ion channel regulation has been established in many other systems (Wu et al, 1998;Melnikova and Gardner, 2001;Lhuillier and Dryer, 2002). The phosphorylated products of these enzymes, including PtdIns[3,4,5]P 3 or PtdIns[3,4]P 2 , can cause direct or indirect activation of a wide variety of intracellular signaling enzymes, many of which contain pleckstrin-homology and FYVE-finger domains that can bind PtdIns[3,4,5]P 3 or PtdIns[3,4]P 2 .…”