Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.

A channel involved in pain perception Voltage-gated sodium (Nav) channels propagate electrical signals in muscle cells and neurons. In humans, Nav1.7 plays a key role in pain perception. It is challenging to target a particular Nav isoform; however, arylsulfonamide antagonists selective for Nav1.7 have been reported recently. Ahuja et al. characterized the binding of these small molecules to human Nav channels. To further investigate the mechanism, they engineered a bacterial Nav channel to contain features of the Nav1.7 voltage-sensing domain that is targeted by the antagonist and determined the crystal structure of the chimera bound to an inhibitor. The structure gives insight into the mechanism of voltage sensing and will enable the design of more-selective Nav channel antagonists. Science , this issue p. 10.1126/science.aac5464

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.

Expression of functional, recombinant ␣7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor ␣7 subunit. In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding. Upon application of 1 mM nicotine, currents were undetectable in HEK293 cells expressing only the ␣7 subunit. In contrast, co-expression of ␣7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous ␣7 receptors. Immunoprecipitation studies demonstrate that ␣7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of ␣7 protein on the plasma membrane of cells lacking ric-3, but surface ␣-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of ␣7 receptors in HEK293 cells. Nicotinic acetylcholine receptors (nAChRs)1 are members of the neurotransmitter-gated ion channel superfamily. They are widely expressed in the central and peripheral nervous system (1) where they influence numerous cellular and physiological processes. At least 17 different genes that code for nAChR subunits have been identified (2, 3), and they assemble as pentamers in different combinations to form a diverse set of nAChR subtypes (4, 5). The simplest case is the homopentameric complex such as that formed by the nAChR ␣7 subunit. The ␣7 receptor, for which ␣-bungarotoxin (␣-Bgt) is a specific and high affinity antagonist, is one of the most abundant receptor subtypes in the mammalian brain (6, 7). The high Ca 2ϩ permeability of the ␣7 receptor (8) suggests an involvement in the activation of Ca 2ϩ -dependent events in neurons such as transmitter release, participation in signal transduction, and a variety of modulatory effects (9). In addition, ␣7 receptors have been implicated in a number of diseases such as schizophrenia, Alzheimers, and Parkinsons disease (1, 10 -12).Heterologous expression of the ␣7 subunit in Xenopus oocytes results in homooligomeric, ␣-Bgt-sensitive receptors that activate and inactivate quickly and are highly permeable to Ca 2ϩ (8,13,14), similar to the properties of ␣7 nAChRs in neuronal cells. Although there have been reports of successful functional expression in some mammalian cell lines (15-18), measurable levels of functional receptors have been difficult to achieve in multiple cell types and this phenomenon appears to be host-cell dependent (19). The reasons for poor heterologous surface expression in these cells are not well understood. Strategies to increase the number of functional receptors on the cell...

Changes in stimulation rate alter the electrical and mechanical characteristics of myocardial cells. We have investigated the possibility that intracellular sodium activity (aiNa) changes with stimulation and correlates with changes in contraction strength. Two kinds of liquid membrane Na+-selective microelectrodes were used to measure aiNa in guinea pig and sheep ventricular muscle and in sheep Purkinje strands. Stimulation produced a rate- and time-dependent elevation of aiNa. Small increases in aiNa were seen at stimulation rates as slow as 0.2 Hz, and faster rates of stimulation elevated aiNa by over 30%. The changes seen in Purkinje strands and ventricular muscle were similar. Following a period of stimulation, aiNa and Vm returned to their pre-stimulus levels with the same time courses. This is consistent with the suggestion that the post-stimulation hyperpolarization is the result of an increased rate of electrogenic Na+ extrusion. The effects of stimulation on aiNa and tension were compared with those of ouabain. The comparison suggests that rapid stimulation could produce increased contraction strength as the result of a substantial gain in intracellular calcium via a Na-Ca exchange mechanism, but that this is only one of several factors determining the force-frequency relationship.