Regulation of hyperpolarization-activated HCN channels by PI(4,5)P2, PLC-linked receptors, and TRIP8b.

摘要

Ionic currents produced by the HCN hyper-polarization-activated cation channels (Ih) are critical for the rhythmic activities of the heart and brain, as well as other cellular functions, such as sensory perception, neuronal integration, and the determination of resting membrane potential and cellular excitability. Previous studies have demonstrated that various signal transduction cascades directly regulate HCN channels. In the best-characterized action, binding of cyclic nucleotides to the cytoplasmic C-terminal cyclic nucleotide-binding domain enhances the opening of HCN channels. More recent evidence suggests the channels may also be regulated by src tyrosine kinase-mediated phosphorylation. In addition to regulation by these signaling events, the HCN channels are also regulated by interactions between the channel and other proteins. However, several lines of evidence suggest that our knowledge of HCN regulation is incomplete. In this thesis, I identify three novel mechanisms of HCN channel regulation that potently regulate the voltage-dependent activation of HCN channels as well as their gating kinetics and levels of current expression.; The voltage-dependence of activation of HCN channels is shifted in inside-out patches by -40 to -60 mV relative to activation in intact cells, a phenomenon referred to as rundown. Less than 20 mV of this hyperpolarizing shift can be due to the influence of the canonical modulator of HCN channels, cAMP. Here, I study the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2) in HCN channel rundown, as hydrolysis of PI(4,5)P2 by lipid phosphatases is thought to underlie rundown of several other channels. I find that bath application of exogenous PI(4,5)P2 reverses the effect of rundown, producing a large, depolarizing shift in HCN2 activation. Several lines of evidence suggest that HCN channels are also regulated by endogenous PI(4,5)P2: (1) Rundown in enhanced by application of an alpha-PIP2 antibody that further depletes PI(4,5)P 2 from the membrane. (2) Rundown is slowed by inhibition of phosphatase activity. (3) Rundown is partially reversed by phosphorylation mediated by membrane-bound phosphatidylinositol kinases.; To test whether neurotransmitter or hormonal signals can dynamically modulate HCN channels by depleting membrane PI(4,5)P2 (as it has been shown for other ion channels regulated by PI(4,5)P2), I monitored HCN channel function following the activation of several cell surface receptors coupled to phospholipase C. Depletion of PI(4,5)P2 is expected to result in a hyperpolarizing shift in the voltage-dependence of HCN activation. In contrast, receptor activation in Xenopus oocytes results in a depolarizing shift in the activation curve of HCN channels. Modulation is preserved in mutants of HCN2 that do not bind cAMP; thus, the modulation cannot be due to changes in cAMP levels. Furthermore, the shift in the voltage-dependence of activation requires PLC activity, but neither IP3-mediated Ca 2+ release nor DAG-dependent kinase activation are required. Importantly, the modulation of HCN2 by PLC-coupled receptors is blocked by inhibitors of phosphatidylinositol kinases, suggesting that the modulation may be due to an increase in PI(4,5)P2 synthesis that is triggered by the PLC-generated signaling cascade, as has been described in other systems.; Finally, I examine the role of protein-protein interactions in HCN regulation. In particular I survey the role of alternate splice forms of TRIP8b, a cytoplasmic protein that is coexpressed with HCN channels in the brain and has previously been shown to strongly internalize HCN channels in both Xenopus oocytes and HEK293 cells. I find that expression of HCN1 with alternate splice forms of TRIP8b result in dramatic and opposing changes in current expression, ranging from a reduction in Ih to undetectable levels with one isoform to a four-fold increase in current with another isoform. Moreover, all TRIP8b isoforms alter HCN1 voltage gating