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Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

Delayed-rectifier K؉ currents (I DR ) in pancreatic ␤-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltagegated K ؉ channel, K V 2.1, is expressed in ␤-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I DR in rodent ␤-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ϳ1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to ␤-cell physiology. In mouse ␤-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the ␤-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the ␤-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

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Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110 -360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing K i ), and those away from the active site enhance catalysis (by increasing k cat /K M ). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively dictated by those of the double mutants.Analyses of mutational effects in the human immunodeficiency virus type-1 (HIV-1) 1 provirus have revealed that as few as four amino acid side chain substitutions, M46I/L63P/V82T/ I84V (4X), in the protease suffice to yield a viral variant crossresistant to a panel of protease inhibitors in clinical studies (1). Three of these inhibitors, Ritonavir (Ro 31-8959), Saquinavir (ABT-538), and Indinavir (MK-639), have been approved by the U. S. Federal Drug Administration as therapeutic agents for the treatment of HIV infection and AIDS. The 4X mutant protease is a poor enzyme. Two of its mutations, V82T and I84V, are located in the active site of the protease (see Fig. 1), and their perturbations of binding have been deduced from the x-ray structure (2); the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site, and the I84V substitution creates a void (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. The combined contribution accounts for a total loss of ϳ3 kcal/mol in energetics. 2 The other two mutations are in the flap (M46I) and hinge (L63P) domains of the protease, and their role in drug resistance is not apparent from the crystallographic data. These changes only induce minor perturbations in the flap domain, as well as in the hinge region, of the native enzyme. One possibility is that the M46I and L63P substitutions affect the stability and/or activity of the enzyme unrelated directly to but ameliorate for the deleterious effect on equilibrium binding by the V82T/I84V mutations.The 4X mutant is derived from clinical studies of AIDS patient viral isolates and is not an intermediate found in the path of emergence of HIV re...