Christopher A. Ahern

Focused Electric Field across the Voltage Sensor of Potassium Channels

Voltage-gated ion channels respond to changes in membrane potential by movement of their voltage sensors across the electric field between cytoplasmic and extracellular solutions. The principal voltage sensors in these proteins are positively charged S4 segments. The absolute magnitude of S4 movement discriminates two competing classes of gating models. In one class, the movement is <10 Angstrom due to the fact that the electric field is focused by aqueous crevices in the channel protein. In an alternative model, based in part on the crystal structure of a potassium channel, the side chains of S4 arginines move their charges across the bilayers electric field, a distance of >25 Angstrom. Here, using tethered charges attached to an S4 segment, we provide evidence that the electric field falls across a distance of <4 Angstrom, supporting a model in which the relative movement between S4 and the electric field is very small.

Electrostatic Contributions of Aromatic Residues in the Local Anesthetic Receptor of Voltage-Gated Sodium Channels

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 &pgr; electrons of the aromatic rings of these residues, namely a cation-&pgr; 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-&pgr; energetics for 1 residue only: Phe1579 in rNaV1.4. 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 &pgr; 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.

Specificity of charge-carrying residues in the voltage sensor of potassium channels.

Positively charged voltage sensors of sodium and potassium channels are driven outward through the membranes electric field upon depolarization. This movement is coupled to channel opening. A recent model based on studies of the KvAP channel proposes that the positively charged voltage sensor, christened the “voltage-sensor paddle”, is a peripheral domain that shuttles its charged cargo through membrane lipid like a hydrophobic cation. We tested this idea by attaching charged adducts to cysteines introduced into the putative voltage-sensor paddle of Shaker potassium channels and measuring fractional changes in the total gating charge from gating currents. The only residues capable of translocating attached charges through the membrane-electric field are those that serve this function in the native channel. This remarkable specificity indicates that charge movement involves highly specialized interactions between the voltage sensor and other regions of the protein, a mechanism inconsistent with the paddle model.

Investigating the Putative Glycine Hinge in Shaker Potassium Channel

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

Modulation of the Cardiac Sodium Channel NaV1.5 by Fyn, a Src Family Tyrosine Kinase

Dynamic modulation of ion channels can produce dramatic alterations of electrical excitability in cardiac myocytes. This study addresses the effects of the Src family tyrosine kinase Fyn on NaV1.5 cardiac sodium channels. Sodium currents were acquired by whole cell recording on HEK-293 cells transiently expressing NaV1.5. Acute treatment of cells with insulin caused a depolarizing shift in steady-state inactivation, an effect eliminated by the Src-specific tyrosine kinase inhibitor PP2. Sodium channels were coexpressed with either constitutively active (FynCA) or catalytically inactive (FynKD) variants of Fyn. FynCA caused a 10-mV depolarizing shift of steady-state inactivation compared with FynKD without altering the activation conductance-voltage relationship. Comparable effects of these Fyn variants were obtained with whole-cell and perforated-patch recording. Tyrosine phosphorylation of immunoprecipitated NaV1.5 was increased in cells expressing FynCA compared with FynKD. We show that Fyn is present in rat cardiac myocytes, and that NaV1.5 channels from these myocytes are tyrosine-phosphorylated. In HEK-293 cells the effect of FynCA on NaV1.5 inactivation is abolished by the single point mutation Y1495F, a residue located within the cytoplasmic linker between the third and fourth homologous domains of the sodium channel. We provide evidence that this linker is a substrate for Fyn in vitro, and that Y1495 is a preferred phosphorylation site. These results suggest that cardiac sodium channels are physiologically relevant targets of Src family tyrosine kinases.

Stirring up controversy with a voltage sensor paddle

Neurons owe their exquisite electrical excitability to voltage-gated ion channels. By creating and shaping the action potential, these voltage-sensitive proteins supply the neuron with crucial communication skills. A steady stream of experimental results, arising from numerous laboratories and employing a diverse repertoire of techniques, has produced a consensus model of the way voltage-gated ion channels sense and respond to changes in membrane potential. In contrast to this consensus mechanism, recent studies of the voltage-gated K(+) channel KvAP suggest a strikingly different mode of action. In this review, these disparate models are compared and critically discussed.

Crystallographic basis for calcium regulation of sodium channels

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca2+-bound calmodulin (Ca2+/CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (NaV1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca2+/CaM binding, which, in turn, sensitize or abolish Ca2+ regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca2+/CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca2+/CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.

Contributions of counter-charge in a potassium channel voltage-sensor domain

Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly co-evolved acidic and aromatic side-chains assist the transfer of cationic side-chains across the transmembrane electric field during voltage-sensing. We investigated the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage-sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side-chains in transmembrane segments S2 and S3, Glu293 and Asp316 in Shaker potassium channels, have little functional effect on conductance-voltage relationships, although Glu293 appears to catalyze S4 movement. Our results suggest that neither Glu293 nor Asp316 engages in electrostatic state-dependent charge-charge interactions with S4, likely because they occupy, and possibly help create, a water-filled vestibule.

The hitchhiker’s guide to the voltage-gated sodium channel galaxy

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.

A Cation-π Interaction Discriminates among Sodium Channels That Are Either Sensitive or Resistant to Tetrodotoxin Block

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 Tyr401 of NaV1.4 through a cation-π interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-π interaction, increased fluorination of Phe401, 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 ∼50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-π 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-π interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels.