Growth Factors Mobilize Multiple Pools ofKCaChannels in Developing Parasympathetic Neurons: Role of ADP-Ribosylation Factors and Related Proteins

Abstract

In developing ciliary ganglion (CG) neurons, movement of functional large-conductance (BK type) Ca2+-activated K+( KCa) channels to the cell surface is stimulated by the endogenous growth factors TGFβ1 and β-neuregulin-1 (NRG1). Here we show that a brief NRG1 treatment (0.5–1.5 h) mobilizes KCachannels in a post-Golgi compartment, but longer treatments (>3.5 h) mobilize KCachannels located in the endoplasmic reticulum or Golgi apparatus. Specifically, the effects of 3.5 h NRG1 treatment were completely blocked by treatments that disrupt Golgi apparatus function. These include inhibition of microtubules, or inhibition of the ADP-ribosylation factor-1 (ARF1) system by brefeldin A, by over-expression of dominant-negative ARF1, or over-expression of an ARF1 GTPase-activating protein that blocks ARF1 cycling between GTP- and GDP-bound states. These treatments had no effect on stimulation of KCaevoked by 1.5 h treatment with NRG1, indicating that short-term responses to NRG1 do not require an intact Golgi apparatus. By contrast, both the acute and sustained effects of NRG1 were inhibited by treatments that block trafficking processes that occur close to the plasma membrane. Thus mobilization of KCawas blocked by treatments than inhibit ADP-ribosylation factor-6 (ARF6) signaling, including overexpression of dominant-negative ARF6, dominant-negative ARNO, or dominant-negative phospholipase D1. TGFβ1, the effects of which on KCaare much slower in onset, is unable to selectively mobilize channels in the post-Golgi pool, and its effects on KCaare completely blocked by inhibition of microtubules, Golgi function and also by plasma membrane ARF6 and phospholipase D1 signaling.