Jon T. Sack

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

The Kv2.1 delayed rectifier potassium channel exhibits high‐level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity‐dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1−/−) mice lacking this channel. Kv2.1−/− mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1−/− mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1−/− mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1−/− animals. Field recordings from hippocampal slices of Kv2.1−/− mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1−/− mice, long‐term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1−/− mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1−/− mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1−/− mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Na+ channel function, regulation, structure, trafficking and sequestration.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na+ channel function and regulation, Na+ channel structure and function, and Na+ channel trafficking, sequestration and complexing.

How to validate a heteromeric ion channel drug target: assessing proper expression of concatenated subunits.

Native subtypes of K+ and other ion channels are often hetero-oligomeric combinations of protein subunits. Drugs that selectively target relevant in vivo heteromers are valuable research tools and potential therapeutics. To recreate heteromeric channels as authentic drug targets, multiple subunits

Concatemers of brain Kv1 channel α subunits that give similar K+ currents yield pharmacologically distinguishable heteromers

At least five subtypes of voltage-gated (Kv1) channels occur in neurons as tetrameric combinations of different alpha subunits. Their involvement in controlling cell excitability and synaptic transmission make them potential targets for neurotherapeutics. As a prerequisite for this, we established herein how the characteristics of hetero-oligomeric K(+) channels can be influenced by alpha subunit composition. Since the three most prevalent Kv1 subunits in brain are Kv1.2, 1.1 and 1.6, new Kv1.6-1.2 and Kv1.1-1.2 concatenated constructs in pIRES-EGFP were stably expressed in HEK cells and the biophysical plus pharmacological properties of their K(+) currents determined relative to those for the requisite homo-tetramers. These heteromers yielded delayed-rectifier type K(+) currents whose activation, deactivation and inactivation parameters are fairly similar although substituting Kv1.1 with Kv1.6 led to a small negative shift in the conductance-voltage relationship, a direction unexpected from the characteristics of the parental homo-tetramers. Changes resulting from swapping Kv1.6 for Kv1.1 in the concatemers were clearly discerned with two pharmacological agents, as measured by inhibition of the K(+) currents and Rb(+) efflux. alphaDendrotoxin and 4-aminopyridine gave a similar blockade of both hetero-tetramers, as expected. Most important for pharmacological dissection of channel subtypes, dendrotoxin(k) and tetraethylammonium readily distinguished the susceptible Kv1.1-1.2 containing oligomers from the resistant Kv1.6-1.2 channels. Moreover, the discriminating ability of dendrotoxin(k) was further confirmed by its far greater ability to displace (125)I-labelled alphadendrotoxin binding to Kv1.1-1.2 than Kv1.6-1.2 channels. Thus, due to the profiles of these two channel subtypes being found to differ, it seems that only multimers corresponding to those present in the nervous system provide meaningful targets for drug development.

A novel epileptic encephalopathy mutation in KCNB1 disrupts Kv2.1 ion selectivity, expression, and localization

A missense mutation in the pore-forming α subunit of a delayed rectifier Kv channel is associated with epileptic encephalopathy, alters the cation selectivity of voltage-gated currents, and disrupts channel expression and localization.

Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels

Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments. DOI: http://dx.doi.org/10.7554/eLife.06774.001

Binding of a Gating Modifier Toxin Induces Intersubunit Cooperativity Early in the Shaker K Channel's Activation Pathway

Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shakers activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions.

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention. Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.