The voltage-gated ion channels and their structural relatives are a large superfamily of membrane proteins specialized for electrical signaling and ionic homeostasis (1). Voltage-gated sodium channels are responsible for the increase in sodium permeability that initiates action potentials in electrically excitable cells (2) and are the molecular target for several groups of neurotoxins, which bind to different receptor sites and alter voltage-dependent activation, conductance, and inactivation (3, 4). Sodium channels are composed of one pore-forming ␣ subunit of ϳ2000 amino acid residues associated with one or two smaller auxiliary subunits, ␤1-␤4 (5-7). The ␣ subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6), and a re-entrant pore loop (P) between S5 and S6 (5). The S4 transmembrane segments are positively charged and serve as voltage sensors to initiate channel activation (8 -14). However, the molecular mechanism of voltage sensing by sodium channels and the other members of the voltage-gated ion channel family is unknown.The initial "sliding helix" (9) or "helical screw" (15) models for voltage sensing proposed that the S4 segments, which have positively charged amino acids at intervals of three residues, transport gating charges outward to activate sodium channels in response to depolarization by moving along a spiral pathway through the protein structure. This movement would preserve interactions with surrounding hydrophilic and negatively charged amino acid residues during gating and thereby stabilize the gating charges in the intramembrane environment. Many structure-function studies have supported this general model (see "Discussion"). In contrast, x-ray crystallographic studies of a bacterial voltage-gated K ϩ channel in complex with detergent and a site-directed antibody yielded a structure in which the S3 and S4 segments lay along the position of the intracellular surface of the membrane, dissociated from the remainder of the protein (16 -18). These results led to the concept that the voltage sensors function as loosely linked "paddles," pivoting through the phospholipid surrounding the core of the ion channel as a semi-rigid body rather than moving gating charge outward through the protein structure. This paddle model makes strikingly different predictions for polypeptide toxins that modify gating by interaction with the voltage sensors. Whereas polypeptide toxins might be able to bind the extracellular end of the voltage sensors in the resting state in a sliding helix or helical screw gating model, the S4 segments would not be expected to be available for toxin binding in the resting state in the paddle model.Scorpion venoms contain two groups of polypeptides toxins that alter sodium channel gating. The ␣-scorpion toxins, as well as sea anemone toxins and some spider toxins, bind to neurotoxin receptor site 3 and slow or block inactivation (19 -22). Amino acid residues that contribute to neurotoxin receptor site

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

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A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.