The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts.

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement.

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

It is now well established that the voltage-sensing S4 segment in voltage-dependent ion channels undergoes a conformational change in response to varying membrane potential. However, the magnitude of the movement of S4 relative to the membrane and the rest of the protein remains controversial. Here, by using histidine scanning mutagenesis in the Shaker K channel, we identified mutants I241H (S1 segment) and I287H (S2 segment) that generate inward currents at hyperpolarized potentials, suggesting that these residues are part of a hydrophobic plug that separates the water-accessible crevices. Additional experiments with substituted cysteine residues showed that, at hyperpolarized potentials, both I241C and I287C can spontaneously form disulphide and metal bridges with R362C, the position of the first charge-carrying residue in S4. These results constrain unambiguously the closedstate positions of the S4 segment with respect to the S1 and S2 segments, which are known to undergo little or no movement during gating. To satisfy these constraints, the S4 segment must undergo an axial rotation of Ϸ180°and a transmembrane (vertical) movement of Ϸ6.5 Å at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field.gating current ͉ metal bridge ͉ omega current ͉ S-S bridge V oltage-gated channels are crucial players in excitability and cell homeostasis. Voltage-dependent sodium and potassium conductances are responsible for the generation and propagation of the nerve impulse (1). The salient property of these channels is the steep membrane potential dependence of their open probability. This voltage dependence is conferred by the voltage sensor that has been identified with their first four transmembrane segments (S1-S4). It is now clear that the four most extracellular basic residues of the S4 segment and the most intracellular acidic residue in the S2 segment in the Shaker K channel are the gating charge-carrying residues that move in the field in response to changes in membrane potential (2, 3). The movement and conformational change of segments S1-S4 within the voltage-sensing module in response to membrane depolarization is somehow coupled to the intracellular gate of the pore domain (S5-S6) to open and close the channel. A multitude of biophysical experiments have attempted to delineate the conformation of the voltage sensor and the position of its charges in the open and closed states. The structural restraints deduced from those experiments have subsequently been shown to be consistent with the overall conformation of the open state from the crystallographic structure of the Kv1.2 channel. However, the experiments providing structural constraints about the closed state conformation of the voltage sensor are more uncertain. Although it is generally accepted that the S1 and S2 helical segments do not move extensively upon gating (4-8), there is disagreement concerning the magnitude of movements associated with the S3 and S4 segments. For exampl...

Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to 'gating currents' of voltage-gated channels. The gating charge-voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

SummaryThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.