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Kenton J Swartz

Publications and source records attributed to Kenton J Swartz.

17 recordsLinked to original sources
PubMed2006

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Animals↗
PubMed2006

Tarantula toxins interacting with voltage sensors in potassium channels.

Voltage-activated ion channels open and close in response to changes in membrane voltage, a process that is crucial for electrical signaling in the nervous system. The venom from many poisonous creatures contains a diverse array of small protein toxins that bind to voltage-activated channels and modify the gating mechanism. Hanatoxin and a growing number of related tarantula toxins have been shown to inhibit activation of voltage-activated potassium (K(v)) channels by interacting with their voltage-sensing domains. This review summarizes our current understanding of the mechanism by which these toxins alter gating, the location of the toxin receptor within K(v) channels and the disposition of this receptor with respect to the lipid membrane. The conservation of tarantula toxin receptors among voltage-activated ion channels will also be discussed.

Amino Acid Sequence↗
PubMed2005

An inhibitor of TRPV1 channels isolated from funnel Web spider venom.

Capsaicin receptor channels (TRPV1) are nonselective cation channels that integrate multiple noxious stimuli in sensory neurons. In an effort to identify new inhibitors of these channels we screened a venom library for activity against TRPV1 channels and found robust inhibitory activity in venom from Agelenopsis aperta, a north American funnel web spider. Fractionation of the venom using reversed-phase HPLC resulted in the purification of two acylpolyamine toxins, AG489 and AG505, which inhibit TRPV1 channels from the extracellular side of the membrane. The activity of AG489 was characterized further, and the toxin was found to inhibit TRPV1 channels with a K(i) of 0.3 microM at -40 mV. Inhibition of TRPV1 channels by AG489 is strongly voltage-dependent, with relief of inhibition at positive voltages, consistent with the toxin inhibiting the channel through a pore-blocking mechanism. We used scanning mutagenesis throughout the TM5-TM6 linker, a region thought to form the outer pore of TRPV1 channels, to identify pore mutations that alter toxin affinity. Four mutants dramatically decrease toxin affinity and several mutants increase toxin affinity, consistent with the notion that the TM5-TM6 linker forms the outer vestibule of TRPV1 channels and that AG489 is a pore blocker.

Agatoxins↗
PubMed2005

Voltage-sensor activation with a tarantula toxin as cargo.

The opening and closing of voltage-activated Na+, Ca2+ and K+ (Kv) channels underlies electrical and chemical signalling throughout biology, yet the structural basis of voltage sensing is unknown. Hanatoxin is a tarantula toxin that inhibits Kv channels by binding to voltage-sensor paddles, crucial helix-turn-helix motifs within the voltage-sensing domains that are composed of S3b and S4 helices. The active surface of the toxin is amphipathic, and related toxins have been shown to partition into membranes, raising the possibility that the toxin is concentrated in the membrane and interacts only weakly and transiently with the voltage sensors. Here we examine the kinetics and state dependence of the toxin-channel interaction and the physical location of the toxin in the membrane. We find that hanatoxin forms a strong and stable complex with the voltage sensors, far outlasting fluctuations of the voltage sensors between resting (closed) conformations at negative voltages and activated (open) conformations at positive voltages. Toxin affinity is reduced by voltage-sensor activation, explaining why the toxin stabilizes the resting conformation. We also find that when hanatoxin partitions into membranes it is localized to an interfacial region, with Trp 30 positioned about 8.5 A from the centre of the bilayer. These results demonstrate that voltage-sensor paddles activate with a toxin as cargo, and suggest that the paddles traverse no more than the outer half of the bilayer during activation.

Amino Acid Substitution↗
PubMed2005

Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel.

VSTx1 is a voltage sensor toxin from the spider Grammostola spatulata that inhibits KvAP, an archeabacterial voltage-activated K(+) channel whose X-ray structure has been reported. Although the receptor for VSTx1 and the mechanism of inhibition are unknown, the sequence of the toxin is related to hanatoxin (HaTx) and SGTx, two toxins that inhibit eukaryotic voltage-activated K(+) channels by binding to voltage sensors. VSTx1 has been recently shown to interact equally well with lipid membranes that contain zwitterionic or acidic phospholipids, and it has been proposed that the toxin receptor is located within a region of the channel that is submerged in the membrane. As a first step toward understanding the inhibitory mechanism of VSTx1, we determined the three-dimensional solution structure of the toxin using NMR. Although the structure of VSTx1 is similar to HaTx and SGTx in terms of molecular fold and amphipathic character, the detailed positions of hydrophobic and surrounding charged residues in VSTx1 are very different than what is seen in the other toxins. The amphipathic character of VSTx1, notably the close apposition of basic and hydrophobic residues on one face of the toxin, raises the possibility that the toxin interacts with interfacial regions of the membrane. We reinvestigated the partitioning of VSTx1 into lipid membranes and find that VSTx1 partitioning requires negatively charged phospholipids. Intrinsic tryptophan fluorescence and acrylamide quenching experiments suggest that tryptophan residues on the hydrophobic surface of VSTx1 have a diminished exposure to water when the toxin interacts with membranes. The present results suggest that if membrane partitioning is involved in the mechanism by which VSTx1 inhibits voltage-activated K(+) channels, then binding of the toxin to the channel would likely occur at the interface between the polar headgroups and the hydrophobic phase of the membrane.

Amino Acid Sequence↗
PubMed2005

Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels.

P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration-response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting alpha-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.

Amino Acid Sequence↗
PubMed2004

Stabilizing the closed S6 gate in the Shaker Kv channel through modification of a hydrophobic seal.

The primary activation gate in K+ channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker (Hackos et al., 2002), we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K+ channels.

Animals↗
PubMed2004

Solution structure and functional characterization of SGTx1, a modifier of Kv2.1 channel gating.

SGTx1 is a peptide toxin isolated from the venom of the spider Scodra griseipes that has been shown to inhibit outward K(+) currents in rat cerebellar granule neurons. Although its amino acid sequence is known to be highly (76%) homologous with that of hanatoxin (HaTx), a well-characterized modifier of Kv2.1 channel gating, the structural and functional characteristics of SGTx1 remain largely unknown. Here we describe the NMR solution structure of SGTx1, the mechanism of its interaction with Kv2.1 channels, and its effect on channel activity once bound. The NMR structure of SGTx1 contains a molecular fold closely resembling the "inhibitor cystine knot" of HaTx, which is composed of an antiparallel beta-sheet and four chain reversals stabilized by three disulfide bonds. Functionally, SGTx1 reversibly inhibited K(+) currents in oocytes expressing Kv2.1 channels. Moreover, generation of steady-state activation curves showed that, consistent with other gating modifiers, SGTx1 acted by shifting the activation of the channel to more depolarized voltages. Thus, the surface profile and mechanism of action of SGTx1 are similar to those of HaTx. Still, detailed comparison of SGTx1 with HaTx revealed differences in binding affinity and conformational homogeneity that result from differences in the charge distribution at the binding surface and in the amino acid composition of the respective beta-hairpin structures in the peptides.

Amino Acid Sequence↗
PubMed2004

Towards a structural view of gating in potassium channels.

Voltage-activated cation channels have pores that are selective for K(+), Na(+) or Ca(2+). Neurons use these channels to generate and propagate action potentials, release neurotransmitters at synaptic terminals and integrate incoming signals in dendrites. Recent X-ray and electron microscopy studies of an archaebacterial voltage-activated K(+) (Kv) channel have provided the first atomic resolution images of the voltage-sensing domains in Kv channels. Although these structures are consistent with previous biophysical analyses of eukaryotic channels, they also contain surprises, which have provoked new ideas about the structure and movements of these proteins during gating. This review summarizes our current understanding of these intriguing membrane proteins and highlights the open questions.

Amino Acid Sequence↗
PubMed2004

Molecular surface of tarantula toxins interacting with voltage sensors in K(v) channels.

The venom from spiders, scorpions, and sea anemone contain a rich diversity of protein toxins that interact with ion channel voltage sensors. Although atomic structures have been solved for many of these toxins, the surfaces that are critical for interacting with voltage sensors are poorly defined. Hanatoxin and SGTx are tarantula toxins that inhibit activation of K(v) channels by interacting with each of the four voltage sensors. In this study we set out to identify the active surface of these toxins by alanine-scanning SGTx and characterizing the interaction of each mutant with the K(v)2.1 channel. Examination of the concentration dependence for inhibition identified 15 mutants with little effect on the concentration dependence for toxin inhibition of the K(v)2.1 channel, and 11 mutants that display moderate to dramatic perturbations. Mapping of these results onto the structure of SGTx identifies one face of the toxin where mutations with pronounced perturbations cluster together, and a backside of the toxin where mutations are well tolerated. The active surface of SGTx contains a ring-like assembly of highly polar residues, with two basic residues that are particularly critical, concentrically arranged around a hydrophobic protrusion containing critical aliphatic and aromatic residues. These results identify the active surface of the toxin and reveal the types of side chains that are important for interacting with voltage sensors.

Amino Acid Sequence↗
PubMed2003

Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels.

In voltage-activated potassium (Kv) channels, basic residues in S4 enable the voltage-sensing domain to move in response to membrane depolarization and thereby trigger the activation gate to open. In the X-ray structure of the KvAP channel, the S4 helix is located near the intracellular boundary of the membrane where it forms a "voltage-sensor paddle" motif with the S3b helix. It has been proposed that the paddle is lipid-exposed and that it translocates through the membrane as it activates. We studied the interaction of externally applied Hanatoxin with the voltage-sensor paddle in Kv channels and show that the toxin binds tightly even at negative voltages where the paddle is resting and the channel is closed. Moreover, measurements of gating charge movement suggest that Hanatoxin interacts with and stabilizes the resting paddle. These findings point to an extracellular location for the resting conformation of the voltage-sensor paddle and constrain its transmembrane movements during activation.

Animals↗
PubMed2003

Constitutive activation of the Shaker Kv channel.

In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521-532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.

Amino Acid Sequence↗
PubMed2003

Defining the conductance of the closed state in a voltage-gated K+ channel.

The opening and closing of the ion conduction pathway in ion channels underlies the generation and propagation of electrical signals in biological systems. Although electrophysiological approaches to measuring the flow of ions in the open state have contributed profoundly to our understanding of ion permeation and gating, it remains unclear how much the ion-throughput rate decreases upon closure of the ion conduction pore. To address this fundamental question, we expressed the Shaker Kv channel at high levels and then measured macroscopic K+ currents at negative membrane voltages and counted the number of channels by quantifying the translocation of gating charge. Our results show that the conductance of the closed state is between 0 and 0.16 fS, or at least 100,000 times lower than for the open state of the channel, indicating that the flow of ions is very tightly regulated in this class of K+ channels.

Animals↗
PubMed2002

Scanning the intracellular S6 activation gate in the shaker K+ channel.

In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed state.

Amino Acid Sequence↗