Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel nonopioid analgesics, such as subtype-selective sodium channel blockers. -Conotoxin KIIIA is representative of -conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ϳ20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na V 1.2 and Na V 1.6 were strongly blocked; within experimental wash times of 40 -60 min, block was reversed very little for Na V 1.2 and only partially for Na V 1.6. Other isoforms were blocked reversibly: Na V 1.3 (IC 50 8 M), Na V 1.5 (IC 50 284 M), and Na V 1.4 (IC 50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na V 1.2 and Na V 1.4; replacement of Trp-8 resulted in reversible block of Na V 1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na V 1.4 than of Na V 1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na V 1.2 and that further engineering of -conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.Venoms are a rich source of neuroactive compounds that target various ion channels and receptors with exquisite potency and selectivity (1-4). There is a continuing need for more subtype-selective pharmacological agents against sodium channels (5), and cone snail venoms provide a unique pharmacopoeia of diverse sodium channel-targeting toxins, including channel blockers as well as inhibitors of channel inactivation (6 -18). -Conotoxins are short peptides that potently block sodium channels (Table 1). The first -conotoxins to be discovered from venom of Conus snails, GIIIA, GIIIB, GIIIC, and PIIIA, were paralytic in fish and potently inhibited skeletal muscle sodium channels in amphibian and mammalian systems.Recently, a second group of -conotoxins has been identified that, in contrast to previously characterized peptides that targeted the skeletal muscle sodium channels, inhibited TTX-resistant (TTX-r) 4 sodium channels when screened on amphibian neuronal preparations (19 -21). This group of conotoxins includes -conotoxin SmIIIA from Conus stercusmuscarum and -conotoxin KIIIA from Conus kinoshitai (Fig. 1). Structural and functional studies on peptides in this group to date suggest that amino acid residues in the C-terminal region of these peptides, including Trp and His (see Table 1), are important for function (19,22).It ...

Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by ␤-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells. mitochondria ͉ permeability transition ͉ polyphosphate ͉ ␤-amyloid peptide ͉ necrosis T he chemical and physical properties of polyphosphate (polyP), including its high negative charge and its ability to form complexes with Ca 2ϩ and to form high energy bonds, underlie its potential to play an important role in cell metabolism. Significant amounts of polyP have been found in bacteria and in lower eukaryotes. In those organisms, it provides energy storage and a reserve pool of inorganic phosphate, participates in regulation of gene expression, protects cells from the toxicity of heavy metals by forming complexes with them, and participates in channel formation through assembly into complexes with Ca 2ϩ and polyhydroxybutyrate (PHB) (polyP/Ca 2ϩ /PHB complex) (1, 2) and possibly through interaction with channelforming proteins (3).PolyP has also been found in all higher eukaryotic organisms tested, where it is localized in various subcellular compartments, including mitochondria (4). Furthermore, mitochondrial polyP can form polyP/Ca 2ϩ /PHB complexes (5) with ion-conducting properties similar to those of native mitochondrial permeability transition pore (mPTP) (6). mPTP opening or formation in the mitochondrial inner membrane is believed to underlie the Ca 2ϩ -induced permeability transition (PT), a phenomenon that causes inner membrane depolarization and disruption of ATP synthesis and plays a central role during various types of necrotic and apoptotic cell death (7). The molecular composition of the conducting pathway of mPTP is currently not well defined.Recently, we have raised the possibility that, in vivo, the polyP/ Ca 2ϩ /PHB complex might comprise the ion-conducting part of the mPTP complex (6). If so, mitochondrial polyP should be essential for mPTP opening/formation. Here, we examine the involvement of polyP in normal mitochondrial function and in PT development during stress. To this end, we specifically reduced levels of mitocho...