Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors.

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.

Unitary recordings of TRP and TRPL channels from isolated Drosophila retinal photoreceptor rhabdomeres: activation by light and lipids.

Transient receptor potential (TRP) channels play key roles in sensory transduction. The TRP family founding members, the Drosophila light-dependent channels, were previously studied under voltage clamp, but had not been characterized in intact rhabdomeres at single-channel level. We report patch-clamp recordings from intact isolated photoreceptors of wt and mutant flies lacking TRP (trp(343)), TRPL (trpl(302)), or both channels (trp(313); trpl(302)). Unitary currents were activated by light in rhabdomere-attached patches. In excised rhabdomeral patches, the channels were directly activated by molecules implicated in phototransduction, such as diacylglycerol and polyunsaturated fatty acids. Currents recorded from trpl photoreceptors are blocked by external Ca(2+), Mg(2+) (1 mM), and La(3+) (20 muM), whereas those from trp photoreceptors are not. Rhabdomeric patches lacked voltage-dependent activity. Patches from trp;trpl mutants were devoid of channels. These characteristics match the macroscopic conductances, suggesting that the unitary currents from Drosophila trpl and trp photoreceptors correspond to TRP and TRPL.

Modulation of human GABArho1 receptors by taurine.

A study was made of the effects of taurine on GABArho1 receptors expressed in Xenopus oocytes. The EC(50) and reversal potentials for GABA, taurine and glycine currents were 2.3+/-0.4 microM (-25+/-0.9 mV), 5+/-0.8mM (-27+/-0.4 mV) and 7+/-0.5mM (-22+/-0.6 mV), respectively. Co-application of GABA and taurine, revealed a taurine concentration-dependent biphasic-modulation of the receptor: at 0.3-30 microM taurine potentiated the GABA-currents, whereas at 0.3-30 mM the GABA-currents were reduced. In contrast glycine potentiated the GABA-currents at all concentrations tested. TPMPA, a GABA(C) specific receptor antagonist, also blocked effectively and reversibly the taurine and glycine currents. Finally, lanthanum and zinc modulated the currents generated by the three amino acids. Taurine is abundant in the retina and our observations suggest that taurine may play an important role modulating the retinal GABAergic transmission.

Protein electrostriction: a possibility of elastic deformation of the alpha-hemolysin channel by the applied field.

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure-Staphylococcus aureus alpha-hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the alpha-hemolysin pore, we analyze the blockage of the channel by the crown/K(+) complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.

Two components of voltage-dependent inactivation in Ca(v)1.2 channels revealed by its gating currents.

Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Ca(v)1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V(1/2)) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge "immobilization," the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li(+) carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations.