Nelly Bennett

Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers

SummaryUnitary currents through cGMP-dependent channels of retinal rods are observed following incorporation into planar lipid bilayers of native vesicles from purified rod outer segment membranes washed free of soluble and peripheral proteins. The influence of the concentration of cGMP, inhibitors (cis-diltiazem, tetracaine and Ag+) and divalent cations (Ca2+, Mg2+, and Co2+) on the conductance and open probability of the channel is described, as well as the voltage dependence of these effects. The cGMP dependence suggests the existence of four binding sites for cGMP and reveals that sequential binding of four cGMP molecules corresponds to the opening of four discrete conductance levels. Finally, we provide conclusive evidence that activated G-protein does not directly inactivate the cGMP-dependent channels of bovine retinal rods.

Vanadate inhibition of the Ca2+‐dependent conformational change of the sarcoplasmic reticulum Ca2+‐ATPase

Vanadate has been shown to be a potent inhibitor of many phosphohydrolases which include a covalent phosphoenzyme intermediate in their enzymatic cycle [ 11. The similar structure of pentavalent vanadium and phosphorus was suggested to be responsible for this inhibitory effect [2]. Extensive work has been done on the inhibitory effect of vanadate on the (Na’,K+)-ATPase [3-S]. Binding to the phosphorylation site is facilitated by K+ and Mg2+ and locks the enzyme in the Ez state [5-61. Vanadate inhibition of Ca2+-ATPase activity of sarcoplasmic reticulum has been reported in [7,8], the apparent affinity for the inhibitor being however much lower than for the __ (Na’,K+)ATPase. These results prompted us to investigate the effect of vanadate on the major conformational change of the Ca2+-ATPase which is induced by Ca2+ binding to the high affinity sites. This change can be followed by intrinsic fluorescence measurements [9]. The main conclusions of this report are: (i) Vanadate binds with high affinity (Kd = 0.2 PM) to the calcium free state E of the Ca’+-ATPase; (ii) The binding is a slow process which requires Mg2+ and is competitively antagonized by orthophosphate; (iii) Binding of vanadate results in stabilizing the calcium free conformation of the Ca2+-ATPase.

Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments. Syntaxin 7 is a SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) protein that is present and active in D. discoideum endosomes [Bogdanovic, Bruckert, Morio and Satre (2000) J. Biol. Chem. 275, 36691-36697]. Here we report the identification of its main SNARE partners by co-immunoprecipitation and MS peptide sequencing. The syntaxin 7 complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)] (where t-SNAREs are SNAREs of the target compartment and v-SNAREs are SNAREs present in donor vesicles). In endosomes and in vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. Antibodies to syntaxin 8 and a soluble recombinant VAMP7 fragment both inhibit in vitro reconstituted D. discoideum endosome fusion. The lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that in endosomes, implying a highly active recycling or retention mechanism. A likely model is that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes, and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming endocytic material.

Gating of retinal rod cation channel by different nucleotides : comparative study of unitary currents

SummarySingle channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K0.5 is about 3 μm for 8Br-cGMP, 18 μm for cGMP and 740 μm for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni2+ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMPor 8Br-cGMP dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry28:2785–2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases Po; addition of cAMP concentrations higher than about 70 μm progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol.123:133–147).

Evidence for differently protonated forms of metarhodopsin II as intermediates in the decay of membrane-bound cattle rhodopsin.

Abstract Suspensions of vesicles from cattle rod disc membranes are flash illuminated at varying pH and temperature; the light induced proton uptake and spectral change associated with the formation of metarhodopsin II are measured. Metarhodopsin II is shown to be diprotonated at 3°C, and to exist in at least two forms at higher temperature: the diprotonated form observed at 3°C(mainly at acid pH), and an unprotonated form (mainly at alkaline pH), which is found to be in temperature dependent equilibrium with metarhodopsin I and/or protonated metarhodopsin II.

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases

We describe a novel protein Ser/Thr phosphatase from Arabidopsis thaliana, PP7, which is only 27‐32% identical in amino acid sequence to the known phosphatases and is the most divergent member of the PPP (PP1/2A/2B) family for today. Some structural features suggest more close relationship of PP7 to the PP5/rdgC subfamily. PP7 contains all of the residues essential for the phosphatase activity and possesses three major insertions in its presumable C‐terminal subdomain, which suggest its unique regulation and/or optimisation of its structure for interaction with specific substrates or regulators. A phosphatase structurally related to PP7 is expressed in rice. PP7 conservation between mono‐ and dicotyledonous plants may point to its essential role in the plant cell.

Effects of cysteine modification on the activity of the cGMP-gated channel from retinal rods

The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide (“activation”) at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments (“inhibition”). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.

Coexpression of α and β Subunits of the Rod Cyclic GMP-Gated Channel Restores Native Sensitivity to Cyclic AMP: Role of D604/N1201

Abstract Coexpression of the βwt and αwt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the β subunit as previously suggested (Varnum et al., 1995, Neuron. 15:619–625), we compared currents from wild-type ( αwt and αwt / βwt ) and from mutated channels ( α D604N, α D604N/ βwt, and αwt/β N1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the β subunit. The D604N mutation in the α subunit and, to a lesser extent, coexpression of the βwt subunit with the αwt subunit reduce the open probability for cGMP compared to that of the αwt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux; Monod et al., 1965, J. Mol. Biol. 12:88–118) suggests that the D604N mutation in the α subunits and coassembly of α and β subunits alter the free energy of gating by cAMP more than that of cAMP binding.

Basis for Intracellular Retention of a Human Mutant of the Retinal Rod Channel a Subunit

A mutant of the a subunit of the retinal rod cyclic GMP-gated channel, [Arg654(1-bp del)], corresponding to a truncated alphaR654Dstop subunit, was previously described in patients with retinitis pigmentosa: when expressed in HEK-293 cells, this mutated a subunit was retained inside the cell, but had normal channel activity in one case where it reached the plasma membrane, indicating that the mechanism of targeting is altered by the mutation, but not the function of the channel. The corresponding mutants of the bovine rod channel (alphaR656D stop), and of the closely related olfactory neuron channel (alphaR632Dstop) alpha subunits were expressed in Xenopus oocytes and their activity was analyzed by patch-clamp. Like their human homologue, these two channels have no activity, and we show that their GFP fusion proteins are accumulated into intracellular compartments. The truncation alone or the R/D mutation alone do not prevent or modify channel activity, indicating that neither the R656 residue nor the C-terminal domain downstream of R656 is necessary for homomeric channel targeting and function. Several mutations of R656 and of the preceding residues in the R656Dstop mutant disclose that the motif responsible for the absence of channel activity is an endoplasmic reticulum retention signal (KXKXXstop) in which the nature of the residues in positions -1 and -4 is determinant.

Protein Ser/Thr phosphatases PPEF interact with calmodulin

Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.