No abstract

Activation of native I H pacemaker channels and channels formed on heterologous expression of some isoforms of their pore forming HCN (hyperpolarization-activated, cyclic nucleotide-regulated) subunits is inhibited by the intravenous general anaesthetic propofol (2,6-diisopropylphenol). Here, we show that inhibition of homomeric HCN1 channels is mediated through anaesthetic association with the membrane embedded channel core, a domain that is highly conserved between this isoform and the relatively insensitive HCN2 and 4 subunits. Decoupling of HCN channel gating from cAMP and internal protons reveals that changes in these second messengers are neither necessary nor sufficient to account for propofol's actions. Modelling of the equilibrium and kinetic behaviour of HCN1 channels in the absence and presence of anaesthetic reveals that (1) gating is best described by models wherein closed and open states communicate via a voltage-independent reaction with no significant equilibrium occupancy of a deactivated open state at non-permissive voltages, and (2) propofol modifies gating by preferentially associating with closed-resting and closed-activated states but a low affinity interaction with the activated open state shapes the effect of the drug under physiological conditions. Our findings illuminate the mechanism of HCN channel gating and provide a framework that will facilitate development of propofol derivates that have altered pharmacological properties and therapeutic potentials.

No abstract

Previously, we have demonstrated a novel interaction between Galpha(o) protein and Purkinje cell protein-2 (Pcp2, also known as L7) in vitro and in transfected cells (Luo and Denker [1999] J. Biol. Chem. 274:10685-10688). Pcp2 is uniquely expressed in cerebellar Purkinje cells and in retinal bipolar neurons, and it may function as a cell-type specific modulator for G protein-mediated cell signaling. This interaction has been further evaluated in the present studies. Coimmunoprecipitation experiments reveal that Pcp2 associates with Galpha(o) in vivo in mouse cerebellum and eye extract. Pcp2 also associate with Galpha(i2) in the cerebellum. No detectable associations of Pcp2 with Galpha(z) and Galpha(q) subunits are observed. The association of Galpha(o) and Pcp2 is detected at postnatal day 1 (P1), and the association remains stable from day 3 (P3) until adulthood. Further, immunofluorescent double labeling and confocal microscopy suggest that Pcp2 and Galpha(o) are colocalized in the distal processes of cerebellar Purkinje cells including axonal endings and dendritic spines. Taken together, these findings indicate colocalization and association of Galpha(o) and Pcp2 in cerebellum and suggest a functional role in regions of synaptic activity.

No abstract