Elisabeth Eismann

Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.

Cyclic nucleotide‐gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage‐dependent Ca2+ blockage of monovalent current in wild‐type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5–pore–S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.

The multi-ion nature of the cGMP-gated channel from vertebrate rods.

1. Native cGMP‐gated channels were studied in rod outer segments of the larval tiger salamander, Ambystoma tigrinum. The alpha‐subunit of the cGMP‐gated channel, here referred to as the wild type (WT), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside‐out configuration and were activated by the addition of 100 or 500 microM cGMP. The current carried by monovalent cations was measured under voltage‐clamp conditions. 2. In the presence of 110 mM Na+ in the extracellular medium and different amounts of Na+ in the intracellular medium, the I‐V relations of the native channel could be described by a single‐site model with a profile of Gibbs free energy with two barriers and a well. A similar result was obtained in the presence of 110 mM Li+ in the extracellular medium and different amounts of Li+ in the intracellular medium. The well depth was 1.4RT (where R is the gas constant and T is the absolute temperature) for both Li+ and Na+. 3. The I‐V relations of the native channel in the presence of 110 mM Na+ on one side of the membrane and 110 mM Li+ on the other side could not be described by the same single‐site model with identical values of barriers and well obtained in the presence of Li+ or Na+ alone: the well for Li+ had to be at least 4RT. 4. In the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane, an anomalous mole fraction effect was observed both in the native and the WT channel. No anomalous behaviour was seen in the presence of Li(+)‐Na+ and Li(+)‐NH4+ mixtures. 5. The anomalous mole fraction effect with mixtures of Li+ and Cs+ was not observed in the channel where glutamate 363 was mutated to a glutamine (E363Q) or an asparagine (E363N). When glutamate 363 was mutated to an aspartate (E363D), the anomalous mole fraction effect with mixtures of Li+ and Cs+ was still observed, although significantly reduced. 6. When lysine 346, arginine 369, aspartate 370 and glutamate 372 were neutralized by mutation to glutamine, the ion permeation through the mutant channels and the WT channel had largely similar properties.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Ligand sensitivity of the α2 subunit from the bovine cone cGMP‐gated channel is modulated by protein kinase C but not by calmodulin

1 Homomeric cyclic nucleotide‐gated (CNG) channels composed of α2 subunits from bovine cone photoreceptors were heterologously expressed in the human embryonic kidney (HEK) 293 cell line. Modulation of cGMP sensitivity by protein kinase C (PKC)‐mediated phosphorylation and by binding of calmodulin (CaM) was investigated in inside‐out patches. 2 A peptide encompassing the putative CaM‐binding site within the N‐terminus of the channel protein binds Ca2+‐CaM with high affinity, yet the ligand sensitivity of α2 channels is not modulated by CaM. 3 PKC‐mediated phosphorylation increased the activation constant (K1/2) for cGMP from 19 to 56 μm and decreased the Hill coefficient (from 2.5 to 1.5). The change in ligand sensitivity involves phosphorylation of the serine residues S577 and S579 in the cGMP‐binding domain. The increase in K1/2 was completely abolished in mutant channels in which the two serine residues were replaced by alanine. 4 An antibody specific for the δ isoform of PKC strongly labels the cone outer segments. 5 Modulation of cGMP affinity of bovine α2 CNG channels by phosphorylation could play a role in the regulation of photoreceptor sensitivity.

Structural Features of Cyclic Nucleotide-Gated Channels

Cyclic nucleotide-gated (CNG) cation channels represent a novel class of ion channels that are directly and cooperatively gated by the binding of guanosine 3’,5’-cyclic monophosphate (cGMP) or adenosi

Cyclic Nucleotide-gated Channels — A Family of Proteins Involved in Vertebrate Photoreception and Olfaction

Cation channels that are directly gated by guanosine 3′,5′-cyclic monophosphate (cGMP) control the flow of ions across the surface membrane of vertebrate rod and cone photoreceptor cells. A similar channel, gated by adenosine 3′,5′-cyclic monophosphate (cAMP) exists in vertebrate olfactory sensory neurons. The channel polypeptide of rod photoreceptors has been identified and the amino acid sequence of the channel polypeptide in rod and olfactory cells has been determined by cloning of cDNA. Although the cyclic nucleotide-gated channels functionally belong to the class of ligand-gated channels, they share some structural features with voltage-gated channels.