Interactions with the physical world are deeply rooted in our sense of touch and depend on ensembles of somatosensory neurons that invade and innervate the skin. Somatosensory neurons convert the mechanical energy delivered in each touch into excitatory membrane currents carried by mechanoelectrical transduction (MeT) channels. Pacinian corpuscles in mammals and touch receptor neurons (TRNs) in Caenorhabditis elegans nematodes are embedded in distinctive specialized accessory structures, have low thresholds for activation, and adapt rapidly to the application and removal of mechanical loads. Recently, many of the protein partners that form native MeT channels in these and other somatosensory neurons have been identified. However, the biophysical mechanism of symmetric responses to the onset and offset of mechanical stimulation has eluded understanding for decades. Moreover, it is not known whether applied force or the resulting indentation activate MeT channels. Here, we introduce a system for simultaneously recording membrane current, applied force, and the resulting indentation in living C. elegans (Feedback-controlled Application of mechanical Loads Combined with in vivo Neurophysiology, FALCON) and use it, together with modeling, to study these questions. We show that current amplitude increases with indentation, not force, and that fast stimuli evoke larger currents than slower stimuli producing the same or smaller indentation. A model linking body indentation to MeT channel activation through an embedded viscoelastic element reproduces the experimental findings, predicts that the TRNs function as a band-pass mechanical filter, and provides a general mechanism for symmetrical and rapidly adapting MeT channel activation relevant to somatosensory neurons across phyla and submodalities.
Abstract-Antiarrhythmics, anticonvulsants, and local anesthetics target voltage-gated sodium channels, decreasing excitability of nerve and muscle cells. Channel inhibition by members of this family of cationic, hydrophobic drugs relies on the presence of highly conserved aromatic residues in the pore-lining S6 segment of the fourth homologous domain of the channel. We tested whether channel inhibition was facilitated by an electrostatic attraction between lidocaine and electrons of the aromatic rings of these residues, namely a cation-interaction. To this end, we used the in vivo nonsense suppression method to incorporate a series of unnatural phenylalanine derivatives designed to systematically reduce the negative electrostatic potential on the face of the aromatic ring. In contrast to standard point mutations at the same sites, these subtly altered amino acids preserve the wild-type voltage dependence of channel activation and inactivation. Although these phenylalanine derivatives have no effect on low-affinity tonic inhibition by lidocaine or its permanently charged derivative QX-314 at any of the substituted sites, high-affinity use-dependent inhibition displays substantial cation-energetics for 1 residue only: Phe1579 in rNa V 1.4. Replacement of the aromatic ring of Phe1579 by cyclohexane, for example, strongly reduces use-dependent inhibition and speeds recovery of lidocaine-engaged channels. Channel block by the neutral local anesthetic benzocaine is unaffected by the distribution of electrons at Phe1579, indicating that our aromatic manipulations expose electrostatic contributions to channel inhibition. These results fine tune our understanding of local anesthetic inhibition of voltage-gated sodium channels and will help the design of safer and more salutary therapeutic agents. Key Words: voltage-gated sodium channels Ⅲ unnatural amino acids Ⅲ local anesthetics Ⅲ cardiac arrhythmias V oltage-gated sodium channels underlie the upstroke and propagation of the action potential in excitable cells of nerve and muscle, making them ideal targets in pharmacological interventions for cardiac arrhythmias, epilepsy, and pain. Antiarrhythmics, anticonvulsants, and local anesthetics comprise a family of sodium channel inhibitors that share chemical and structural similarity. All sodium channel isoforms, including the cardiac channel Na V 1.5, are inhibited by these compounds, 1-3 with differences in sensitivity largely attributed to divergent biophysical aspects of gating kinetics. 3 At physiological pH values, these inhibitors are typically lipid-soluble cations that are highly efficacious in the treatment of hyperexcitability disorders because of the preference of the inhibitors for inactivated channels, a conformational state that is prevalent during high-frequency firing of action potentials. This state-dependent inhibition is rationalized by models that propose that inactivation exposes a high-affinity binding site.A general deficiency of this class of compounds is their lack of specificity. For example, the a...
The founding members of the superfamily of DEG/ENaC ion channel proteins are C. elegans proteins that form mechanosensitive channels in touch and pain receptors. For more than a decade, the research community has used mutagenesis to identify motifs that regulate gating. This review integrates insight derived from unbiased in vivo mutagenesis screens with recent crystal structures to develop new models for activation of mechanically-gated DEGs.
Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation–π interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation–π binding ability of an aromatic ring, progressively increased the inhibitory constant K i for the TEA block of Shaker. A larger increase in K i was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation–π component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation–π mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation–π interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.
Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr 401 of Na V 1.4 through a cation-interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-interaction, increased fluorination of Phe 401 , which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by ϳ50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels.Sodium channels control electrical excitability in muscle and nervous tissues by driving membrane depolarization during an action potential. These large (ϳ260 kDa) proteins have four homologous transmembrane domains that are arranged in a clockwise orientation around a central pore (1). Numerous sodium channel isoforms exist and share the common traits of being exceptionally sensitive to voltage and selective for Na ϩ ions (2). They can, however, differ in expression patterns, inactivation time course, sensitivity to toxins, and effects of auxiliary subunits. In terms of extracellular block by the guanidinium toxin tetrodotoxin (TTX), 2 sodium channels have been deemed binary in their sensitivity; they are either TTX-sensitive, blocked by low nanomolar concentrations, or TTX-resistant with blocking constants in the micromolar range (3). Members of the former class include isoforms expressed in brain (Na V 1.1, Na V 1.2, and Na V 1.3), skeletal muscle (Na V 1.4), and peripheral nervous system (Na V 1.6 and Na V 1.7); the latter class includes isoforms of cardiac muscle (Na V 1.5) and sensory neurons (Na V 1.8 and Na V 1.9). A number of point mutations in the external vestibule of the channel can substantially disrupt TTX binding (4 -8), yet many of these residues are conserved between sensitive and resistant isoforms and therefore cannot explain isoform-specific TTX sensi...
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