A B ST RA C T The effects of external Zn +2 and other divalent cations on K channels in squid giant axons were studied. At low concentration (2 mM) Zn +2 slows opening kinetics without affecting closing kinetics . Higher concentrations (5-40 mM) progressively slow opening and speed channel closing to a lesser degree. In terms of "shifts," opening kinetics are strongly shifted to the right on the voltage axis, and OFF kinetics much less so . The shift of the conductancevoltage relation along the axis is intermediate . Zinc's kinetic effects show little sign of saturation at the highest concentration attainable . Zn does not alter the shape of the instantaneous current-voltage relation of open channels . Some other divalent cations have effects similar to Zn+2, Hg2+ being the most potent and Ca 12 the least. After treatment with H g+2, which is irreversible, Zn +2 still slows opening kinetics, which suggests that each channel has at least two sites for divalent cation action . The results are not compatible with a simple theory of fixed, uniform surface charges. They suggest that external cations interact directly with a negatively charged element of the gating apparatus that moves inward from the membrane's outer surface during activation . Examination of normal kinetics shows that there is a slow step somewhere in the chain leading to channel opening, but the slowest step must not be the last one.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

A B S TRACT The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn 2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e ., the amount of additional depolarization required to overcome the Zn 2+ effect . In these terms the mean shifts caused by 30 mM Zn2+ were +29 .5 mV for Na channel opening (ON) kinetics (hit ON), +2 mV for closing (OFF) kinetics (TOFF), and +8.4 mV for the gN8-V curve. Zn 2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of^-0 .67 . Outward current is unaffected . Effects of Zn 2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNs. ON gating current kinetics are shifted by +27 .5 mV, OFF kinetics by +6 mV, and the Q V distribution by +6.5 mV . Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants . More than one of the several steps in activation must be affected . The results are not compatible with the usual simple theory of uniform fixed surface charge . They suggest instead that Zn 2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation .

Sodium channels in nerve and muscle cells are functionally similar across wide phylogenetic boundaries and are usually thought to represent a single, homogeneous population that initiates the action potential at threshold and unerringly transmits it along the surface membrane. In marked contrast, many cell types are known to have several distinct potassium permeability systems. Distinguishable populations of Na channels have been reported in a few cell types, however, including denervated skeletal muscle, embryonic cardiac muscle, Purkinje cell somata and non-myelinated axons at low temperature. We report here that in squid giant axon, in standard experimental conditions, there are two functionally distinct populations of Na channels. The newly discovered population accounts for only a few per cent of the total Na permeability. The channels are selectively activated by small depolarizations and have very slow closing kinetics. Because these channels activate at voltages near the resting potential and tend to stay open for long times, they must dominate behaviour of the axon membrane in the threshold region for action potential initiation.