We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one ‘pseudo‐operator’ O2 or O3 decreases repression by wild‐type tetrameric Lac repressor approximately 2‐ to 3‐fold; absence of both ‘pseudo‐operators’ decreases repression greater than 50‐fold. O1 alone represses under these conditions only approximately 20‐fold. Dimeric active Lac repressor (iadi) represses the wild‐type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells from the external and internal face. We studied ion selectivity and blockage by Ca2+ of wild-type and mutant channels in a heterologous expression system. ExternalCa2+ blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of -2. The blockage from the internal face requires 1000-fold higher Ca2+ concentrations. Neutralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeabilities nearly unaffected. The current blockage at -80 mV requires =2000-fold higher external Ca2+ concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.The cyclic nucleotide-gated channel of vertebrate rod photoreceptor cells (rod channel) belongs to a class of ligandgated channels that are directly and cooperatively opened by the binding of cyclic nucleotides (cGMP or cAMP) (1-11). The rod channel is cation selective but does not appreciably discriminate between monovalent alkali cations (1,4,(12)(13)(14)(15)(16)(17). Divalent cations also permeate the rod channel and thereby block the Na+ current in a voltage-dependent manner (15-29). Ca2+ entry is of great physiologic importance because Ca2+ is part of a negative feedback mechanism that regulates the recovery of the light response and light adaptation (for review, see refs. 30 and 31). Blockage by both external and internal Ca2+ is voltage dependent, indicating a binding site(s) for Ca2+ within the channel pore (26, 28). Internal Ca2+ decreases both the open probability and the single-channel conductance (32). External Ca2+ appears to reduce singlechannel conductance from 25 to =0.1 pS (33,34).Comparison with the protein sequences of K+ channels has led to the prediction that the pore region of the rod channel is located between transmembrane segments H4 and H5 (refs. 7, 8, and 35-37; for reviews, see refs. 38-41). The pore region of K+ channels contains two adjacent amino acid residues (Y445, G446; Fig. 1) that are missing in the respective region of the rod channel. By deletion of these residues, the Shaker K+ channel is converted into a nonselective cation channel (36). The sensitivity to blockage by divalent cations that is increased in the deletion mutant is controlled by the adjacent acidic residue D447 (36). This residue corresponds to glutamate-363 (E363) in the pore region ofthe rod channel. We demonstrate by heterologous expression of wild-type (wt) and mutant rod channels that E363 is crucial for binding of both monovalent and divalent cations and resides inside the pore lumen. MATERIALS AND METHODSSite-Directed Mutagenesis and in Vitro Transcription. The point mutations K346Q, E36...

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3;Srcyclic monophosphate (CAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.

Cyclic nucleotide-gated ionic channels in pho- (9). The mamnima rod and olfactory cyclic nucleotide-gated channels contain a threonine residue at this particular position (6-8; Fig. 1). It has been proposed that this alanine/threonine difference might have been important in the evolutionary divergence of cyclic nucleotidebinding sites and that it provides the structural basis for discriminating between cAMP and cGMP (9). We tested the validity of this hypothesis for cyclic nucleotide-gated channels by mutagenesis and expression of wild-type and mutant channels from rod photoreceptors and olfactory epithelium. MATERIALS AND METHODSConstruction of Recombinant pCHOLF102. PCR (13) was done with pCHOLF100 (8) as template and the following primers: a 5' adapter primer [containing an EcoRV restriction site, a consensus sequence for eukaryotic ribosomal-binding sites (14), and the first nine nucleotides from the coding region of CHOLF1001 and a gene-specific 3' primer. The EcoRV/DraIII-digested PCR product replaced the corresponding fragment of pCHOLF100 to yield pCHOLF101. The insert of pCHOLF101 was subcloned into a pT7T3 vector to yield pCHOLF102.Site-Directed Mutagenesis. The point mutations at positions 560 and 537 of the rod and olfactory channel polypeptides, respectively, were introduced by PCR procedure (13) with synthetic oligonucleotides containing the desired nucleotide substitutions.Rod-channel mutant T560A was constructed by the method of Hemsley et al. (15). A circular plasmid with the wild-type rod-channel sequence from pRCG1 (6) was amplified by a pair of primers located "back-to-back" on opposite DNA strands. The resulting PCR product was recircularized and digested with Nsi I and Sty I. The corresponding Nsi I-Sty I fragment in pRCG1 was replaced by the mutated fragment to create pT560A. For the construction ofrod-channel mutant T560S, we took advantage of a newly introduced Cla I restriction site near codon 560 in pT560A. A PCR fragment was produced by using a mutagenic and a complementary primer and linearized pT560A as template. The Nsi I-Cla Ifagment containing the mutation was exchanged for the corresponding Nsi I-Cla I fragment ofpT560A to generate pT560S.Both olfactory-channel mutants T537A and T537S were constructed by combining two overlapping PCR fragments with the aid of newly introduced restriction sites at the locus of mutation (BssHII for pT537A and Rsr II for pT537S) and other suitable restriction sites in the plasmid. pCHOLF102 was used as template. All mutations were verified by sequencing of the entire insert with the dideoxynucleotide chain-termination method.Functional Expression. mRNA specific for the rodphotoreceptor channel, the olfactory channel, and the mutant channels was synthesized in vitro (16) by using the respective linearized plasmid cDNA as template. Transcription was primed with the cap dinucleotide 7-methylguanosine(5')-triphospho(5')guanosine (0.6 mM) (17). Macroscopic current measurements on excised inside-out patches (18)(19)(20) were made after injection...

1. Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander, Ambystoma tigrinum. The a-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 /SM 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 10 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 Nae. 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. 7. The results here reported indicate that: (i) the native and the WT cGMP-gated channels are both multi-ion pores; (ii) the mutant channels E363Q and E363N behave as a single-ion pore; (iii) the multi-ion nature of the WT channel is primarily controlled by glutamate 363 and not by other charged residues in the pore region.The ion channel controlled by light in vertebrate rod Tanaka, 1990; Liihring, Hanke, Simmoteit & Kaupp, photoreceptor cells, usually referred to as the cGMP-gated 1990) and is permeable to a variety of divalent cations channel, does not appreciably discriminate between (Colamartino, Menini & Torre, 1991). This channel is monovalent alkali cations (Menini,