Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

https://science.sciencemag.org/content/sci/245/4922/1066.full.pdf

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

/pdf/the-scanning-model-for-translation-an-update-52r2in8l71.pdf

The scanning model for translation: an update.

The family of voltage-gated sodium channels initiates action potentials in all types of excitable cells. Nine members of the voltage-gated sodium channel family have been characterized in mammals, and a 10th member has been recognized as a related protein. These distinct sodium channels have similar structural and functional properties, but they initiate action potentials in different cell types and have distinct regulatory and pharmacological properties. This article presents the molecular relationships and physiological roles of these sodium channel proteins and provides comprehensive information on their molecular, genetic, physiological, and pharmacological properties.

https://pharmrev.aspetjournals.org/content/pharmrev/57/4/411.full.pdf

International Union of Pharmacology. XLVIII. Nomenclature and Structure-Function Relationships of Voltage-Gated Calcium Channels

http://www.columbia.edu/itc/gsas/g9600/2002/misc/Catterall_review.pdf

From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-Gated Sodium Channels

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.

X-ray structure of a voltage-dependent K+ channel

Cloning and sequence analysis of cDNA for the Electrophorus electricus electroplax sodium channel indicate that this protein, consisting of 1,820 amino acid residues, exhibits four repeated homology units, which are presumably oriented in a pseudosymmetric fashion across the membrane. Each homology unit contains a unique segment with clustered positively charged residues, which may be involved in the gating structure, possibly in conjunction with negatively charged residues clustered elsewhere.

Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence.

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials1–3. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel)4,5 and from chick cardiac muscle6 consist of a single polypeptide of relative molecular mass (Mr) ∼260,000 (260K), whereas those purified from rat brain7 and skeletal muscle8 contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37–45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA9. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes10–19. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.

Existence of distinct sodium channel messenger RNAs in rat brain

The voltage-gated sodium channel is a transmembrane protein essential for the generation of action potentials in excitable cells. It has been reported that sodium channels purified from the electric organ of the electric eel, Electrophorus electricus, and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000, whereas those purified from rat brain and from rat and rabbit skeletal muscle contain, in addition to the large polypeptide, one or two smaller polypeptides of Mr 33,000-43,000. The primary structures of the Electrophorus sodium channel and two distinct sodium channel large polypeptides (designated as sodium channels I and II) from rat brain have been elucidated by cloning and sequencing the complementary DNAs. The purified sodium channel preparations from Electrophorus electroplax and from mammalian muscle and brain, when reconstituted into lipid vesicles or planar lipid bilayers, exhibit some functional activities. The successful reconstitution with the Electrophorus preparation would imply that the large polypeptide alone is sufficient to form functional sodium channels. However, studies with the rat brain preparation suggest that the smaller polypeptide of Mr 36,000 is also required for the integrity of the saxitoxin (STX) or tetrodotoxin (TTX) binding site of the sodium channel. Here we report that the messenger RNAs generated by transcription of the cloned cDNAs encoding the rat brain sodium channel large polypeptides, when injected into Xenopus oocytes, can direct the formation of functional sodium channels.

Expression of functional sodium channels from cloned cDNA

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of α2βγδ, and this is thought to be also the case for the mammalian skeletal muscle AChR1–3. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four sub-units of the Torpedo californica electroplax4–6 and calf muscle AChR7–10 and of the α- and γ-subunits of the human muscle AChR7,11; the primary structures of the γ-subunit of the T. californien AChR12 and the α-subunit of the Torpedo marmorata AChR13,14 have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the e-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the γ-subunit than with the other subunits. cDNA expression studies indicate that the calf e-subunit, as well as the calf γ-subunit, can replace the Torpedo γ-subunit to form the functional receptor in combination with the Torpedo α-, β- and δ-subunits.

Takayuki Ikeda

Papers

Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence.

Existence of distinct sodium channel messenger RNAs in rat brain

Expression of functional sodium channels from cloned cDNA

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle.