Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes.

1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.

Modulation by nitric oxide of rat brain GABAA receptors.

The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L-arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells.