Cytotoxic Swelling of Sick Excitable Cells - Impaired Ion Homeostasis and Membrane Tension Homeostasis in Muscle and Neuron.

When they become simultaneously leaky to both Na+ and Cl-, excitable cells are vulnerable to potentially lethal cytotoxic swelling. Swelling ensues in spite of an isosmotic milieu because the entering ions add osmolytes to the cytoplasm's high concentration of impermeant anionic osmolytes. An influx of osmotically-obliged water is unavoidable. A cell that cannot stanch at least one the leaks will succumb to death by Donnan effect. "Sick excitable cells" are those injured through ischemia, trauma, inflammation, hyperactivity, genetically-impaired membrane skeletons and other insults, all of which foster bleb-damage to regions of the plasma membrane. Nav channels resident in damaged membrane exhibit left-shifted kinetics; the corresponding Nav window conductance constitutes a Na+-leak. In cortical neurons, sustained depolarization to ∼-20mV elicits a sustained lethal gCl. Underlying Vrest in skeletal muscle is a constitutively active gCl; not surprisingly therefore, dystrophic muscle fibers, which are prone to bleb damage and which exhibit Nav-leak and Na+-overload, are prone to cytotoxic swelling. To restore viability in cytotoxically swelling neurons and muscle, the imperative of fully functional ion homeostasis is well-recognized. However, as emphasized here, in a healthy excitable cell, fully functional membrane tension homeostasis is also imperative. ATPase-pumps keep plasma membrane batteries charged, and ATPase-motor proteins maintain membrane tone. In sick excitable cells, neither condition prevails.

Calculating the Consequences of Left-Shifted Nav Channel Activity in Sick Excitable Cells.

Two features common to diverse sick excitable cells are "leaky" Nav channels and bleb damage-damaged membranes. The bleb damage, we have argued, causes a channel kinetics based "leakiness." Recombinant (node of Ranvier type) Nav1.6 channels voltage-clamped in mechanically-blebbed cell-attached patches undergo a damage intensity dependent kinetic change. Specifically, they experience a coupled hyperpolarizing (left) shift of the activation and inactivation processes. The biophysical observations on Nav1.6 currents formed the basis of Nav-Coupled Left Shift (Nav-CLS) theory. Node of Ranvier excitability can be modeled with Nav-CLS imposed at varying LS intensities and with varying fractions of total nodal membrane affected. Mild damage from which sick excitable cells might recover is of most interest pathologically. Accordingly, Na+/K+ ATPase (pump) activity was included in the modeling. As we described more fully in our other recent reviews, Nav-CLS in nodes with pumps proves sufficient to predict many of the pathological excitability phenomena reported for sick excitable cells. This review explains how the model came about and outlines how we have used it. Briefly, we direct the reader to studies in which Nav-CLS is being implemented in larger scale models of damaged excitable tissue. For those who might find it useful for teaching or research purposes, we coded the Nav-CLS/node of Ranvier model (with pumps) in NEURON. We include, here, the resulting "Regimes" plot of classes of excitability dysfunction.

Dissemination of a Child Passenger Safety Program Through Trauma Center-Community Partnerships.

Improper child passenger restraint use contributes to higher pediatric motor vehicle collision morbidity and mortality among cultural minority populations. Child passenger safety education improves caregiver knowledge of restraint use, but effective interventions require culturally specific programming. The purpose of this study was to evaluate the effectiveness of a child passenger safety education program culturally adapted through a pediatric trauma center's community partnerships. A nonexperimental observational cohort study using program evaluation data for the child passenger safety education programs during a 24-month period. Paired pretest/posttest self-reported survey responses measured changes in caregiver knowledge and self-efficacy of restraint use. Data were analyzed by class location and by caregiver language using a paired t test and Wilcoxon's signed ranks test. A total of 1,795 paired survey responses were collected in English, Spanish, or Russian. An increase in mean knowledge scores occurred overall, with a difference in mean of 0.565 (SE = 0.022, 95% CI [0.521, 0.607]). Stratification by class site and by language reflected significant increases in median scores, but findings were variable by study group. Pretest median scores for self-efficacy of restraint use were high for all groups, but the increases in posttest medians were also significant across groups (p ≤ .001). Caregiver knowledge and self-efficacy for child passenger restraint use increased after participation in the community classes. The pediatric trauma center's community partnerships facilitated uptake and adaption of the child passenger safety education programs and increased the injury prevention outreach to minority communities.

Membrane order parameters for interdigitated lipid bilayers measured via polarized total-internal-reflection fluorescence microscopy.

Incorporating ethanol in lipid membranes leads to changes in bilayer structure, including the formation of an interdigitated phase. We have used polarized total-internal-reflection fluorescence microscopy (pTIRFM) to measure the order parameter for Texas Red DHPE incorporated in the ethanol-induced interdigitated phase (LßI) formed from ternary lipid mixtures comprising dioleoylphosphatidylcholine, cholesterol and egg sphingomyelin or dipalmitoylphosphatidylcholine. These lipid mixtures have 3 co-existing phases in the presence of ethanol: liquid-ordered, liquid-disordered and LßI. pTIRFM using Texas Red DHPE shows a reversal in fluorescence contrast between the LßI phase and the surrounding disordered phase with changes in the polarization angle. The contrast reversal is due to changes in the orientation of the dye, and provides a rapid method to identify the LßI phase. The measured order parameters for the LßI phase are consistent with a highly ordered membrane environment, similar to a gel phase. An acyl-chain labeled BODIPY-FL-PC was also tested for pTIRFM studies of ethanol-treated bilayers; however, this probe is less useful since the order parameters of the interdigitated phase are consistent with orientations that are close to random, either due to local membrane disorder or to a mixture of extended and looping conformations in which the fluorophore is localized in the polar headgroup region of the bilayer. In summary, we demonstrate that order parameter measurements via pTIRFM using Texas Red-DHPE can rapidly identify the interdigitated phase in supported bilayers. We anticipate that this technique will aid further research in the effects of alcohols and other additives on membranes.

Stimulation-induced ectopicity and propagation windows in model damaged axons.

Neural tissue injuries render voltage-gated Na+ channels (Nav) leaky, thereby altering excitability, disrupting propagation and causing neuropathic pain related ectopic activity. In both recombinant systems and native excitable membranes, membrane damage causes the kinetically-coupled activation and inactivation processes of Nav channels to undergo hyperpolarizing shifts. This damage-intensity dependent change, called coupled left-shift (CLS), yields a persistent or "subthreshold" Nav window conductance. Nodes of Ranvier simulations involving various degrees of mild CLS showed that, as the system's channel/pump fluxes attempt to re-establish ion homeostasis, the CLS elicits hyperexcitability, subthreshold oscillations and neuropathic type action potential (AP) bursts. CLS-induced intermittent propagation failure was studied in simulations of stimulated axons, but pump contributions were ignored, leaving open an important question: does mild-injury (small CLS values, pumps functioning well) render propagation-competent but still quiescent axons vulnerable to further impairments as the system attempts to cope with its normal excitatory inputs? We probe this incipient diffuse axonal injury scenario using a 10-node myelinated axon model. Fully restabilized nodes with mild damage can, we show, become ectopic signal generators ("ectopic nodes") because incoming APs stress Na+ / K+ gradients, thereby altering spike thresholds. Comparable changes could contribute to acquired sodium channelopathies as diverse as epileptic phenomena and to the neuropathic amplification of normally benign sensory inputs. Input spike patterns, we found, propagate with good fidelity through an ectopically firing site only when their frequencies exceed the ectopic frequency. This "propagation window" is a robust phenomenon, occurring despite Gaussian noise, large jitter and the presence of several consecutive ectopic nodes.

Spontaneous excitation patterns computed for axons with injury-like impairments of sodium channels and Na/K pumps.

In injured neurons, "leaky" voltage-gated sodium channels (Nav) underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO), to ectopic (sometimes paroxysmal) excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na(+)-loading and "Nav-CLS", i.e., coupled left-(hyperpolarizing)-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge) results in comparable impairment: left-shifted activation and availability and hence left-shifted I(Na-window). A recent computation study revealed that CLS-based I(Na-window) left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic "smeared" injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons) are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For "mild CLS injury", a prominent feature is slow pump/leak-mediated E(Ion) oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist "device" could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.

Coupled left-shift of Nav channels: modeling the Na⁺-loading and dysfunctional excitability of damaged axons.

Injury to neural tissue renders voltage-gated Na⁺ (Nav) channels leaky. Even mild axonal trauma initiates Na⁺-loading, leading to secondary Ca²âº-loading and white matter degeneration. The nodal isoform is Nav1.6 and for Nav1.6-expressing HEK-cells, traumatic whole cell stretch causes an immediate tetrodotoxin-sensitive Na⁺-leak. In stretch-damaged oocyte patches, Nav1.6 current undergoes damage-intensity dependent hyperpolarizing- (left-) shifts, but whether left-shift underlies injured-axon Nav-leak is uncertain. Nav1.6 inactivation (availability) is kinetically limited by (coupled to) Nav activation, yielding coupled left-shift (CLS) of the two processes: CLS should move the steady-state Nav1.6 "window conductance" closer to typical firing thresholds. Here we simulated excitability and ion homeostasis in free-running nodes of Ranvier to assess if hallmark injured-axon behaviors--Na⁺-loading, ectopic excitation, propagation block--would occur with Nav-CLS. Intact/traumatized axolemma ratios were varied, and for some simulations Na/K pumps were included, with varied in/outside volumes. We simulated saltatory propagation with one mid-axon node variously traumatized. While dissipating the [Na⁺] gradient and hyperactivating the Na/K pump, Nav-CLS generated neuropathic pain-like ectopic bursts. Depending on CLS magnitude, fraction of Nav channels affected, and pump intensity, tonic or burst firing or nodal inexcitability occurred, with [Na⁺] and [K⁺] fluctuating. Severe CLS-induced inexcitability did not preclude Na⁺-loading; in fact, the steady-state Na⁺-leaks elicited large pump currents. At a mid-axon node, mild CLS perturbed normal anterograde propagation, and severe CLS blocked saltatory propagation. These results suggest that in damaged excitable cells, Nav-CLS could initiate cellular deterioration with attendant hyper- or hypo-excitability. Healthy-cell versions of Nav-CLS, however, could contribute to physiological rhythmic firing.

The Donnan-dominated resting state of skeletal muscle fibers contributes to resilience and longevity in dystrophic fibers.

Duchenne muscular dystrophy (DMD) is an X-linked dystrophin-minus muscle-wasting disease. Ion homeostasis in skeletal muscle fibers underperforms as DMD progresses. But though DMD renders these excitable cells intolerant of exertion, sodium overloaded, depolarized, and spontaneously contractile, they can survive for several decades. We show computationally that underpinning this longevity is a strikingly frugal, robust Pump-Leak/Donnan (P-L/D) ion homeostatic process. Unlike neurons, which operate with a costly "Pump-Leak-dominated" ion homeostatic steady state, skeletal muscle fibers operate with a low-cost "Donnan-dominated" ion homeostatic steady state that combines a large chloride permeability with an exceptionally small sodium permeability. Simultaneously, this combination keeps fiber excitability low and minimizes pump expenditures. As mechanically active, long-lived multinucleate cells, skeletal muscle fibers have evolved to handle overexertion, sarcolemmal tears, ischemic bouts, etc.; the frugality of their Donnan dominated steady state lets them maintain the outsized pump reserves that make them resilient during these inevitable transient emergencies. Here, P-L/D model variants challenged with DMD-type insult/injury (low pump-strength, overstimulation, leaky Nav and cation channels) show how chronic "nonosmotic" sodium overload (observed in DMD patients) develops. Profoundly severe DMD ion homeostatic insult/injury causes spontaneous firing (and, consequently, unwanted excitation-contraction coupling) that elicits cytotoxic swelling. Therefore, boosting operational pump-strength and/or diminishing sodium and cation channel leaks should help extend DMD fiber longevity.

Modulation of KvAP unitary conductance and gating by 1-alkanols and other surface active agents.

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.

Mechanosensitive closed-closed transitions in large membrane proteins: osmoprotection and tension damping.

Multiconformation membrane proteins are mechanosensitive (MS) if their conformations displace different bilayer areas. Might MS closed-closed transitions serve as tension buffers, that is, as membrane "spandex"? While bilayer expansion is effectively instantaneous, transitions of bilayer-embedded MS proteins are stochastic (thermally activated) so spandex kinetics would be critical. Here we model generic two-state (contracted/expanded) stochastic spandexes inspired by known bacterial osmovalves (MscL, MscS) then suggest experimental approaches to test for spandex-like behaviors in these proteins. Modeling shows: 1), spandex kinetics depend on the transition state location along an area reaction coordinate; 2), increasing membrane concentration of a spandex right-shifts its midpoint (= tension-Boltzmann); 3), spandexes with midpoints below the activating tension of an osmovalve could optimize osmovalve deployment (required: large midpoint, barrier near the expanded state); 4), spandexes could damp bilayer tension excursions (required: midpoint at target tension, and for speed, barrier halfway between the contracted and expanded states; the larger the spandex Delta-area, the more precise the maintenance of target tension; higher spandex concentrations damp larger amplitude strain fluctuations). One spandex species could not excel as both first line of defense for osmovalve partners and tension damper. Possible interactions among MS closed-closed and closed-open transitions are discussed for MscS- and MscL-like proteins.

Membrane trauma and Na+ leak from Nav1.6 channels.

During brain trauma, white matter experiences shear and stretch forces that, without severing axons, nevertheless trigger their secondary degeneration. In central nervous system (CNS) trauma models, voltage-gated sodium channel (Nav) blockers are neuroprotective. This, plus the rapid tetrodotoxin-sensitive Ca2+ overload of stretch-traumatized axons, points to "leaky" Nav channels as a pivotal early lesion in brain trauma. Direct effects of mechanical trauma on neuronal Nav channels have not, however, been tested. Here, we monitor immediate responses of recombinant neuronal Nav channels to stretch, using patch-clamp and Na+-dye approaches. Trauma constituted either bleb-inducing aspiration of cell-attached oocyte patches or abrupt uniaxial stretch of cells on an extensible substrate. Nav1.6 channel transient current displayed irreversible hyperpolarizing shifts of steady-state inactivation [availability(V)] and of activation [g(V)] and, thus, of window current. Left shift increased progressively with trauma intensity. For moderately intense patch trauma, a approximately 20-mV hyperpolarizing shift was registered. Nav1.6 voltage sensors evidently see lower energy barriers posttrauma, probably because of the different bilayer mechanics of blebbed versus intact membrane. Na+ dye-loaded human embryonic kidney (HEK) cells stably transfected with alphaNav1.6 were subjected to traumatic brain injury-like stretch. Cytoplasmic Na+ levels abruptly increased and the trauma-induced influx had a significant tetrodotoxin-sensitive component. Nav1.6 channel responses to cell and membrane trauma are therefore consistent with the hypothesis that mechanically induced Nav channel leak is a primary lesion in traumatic brain injury. Nav1.6 is the CNS node of Ranvier Nav isoform. When, during head trauma, nodes experienced bleb-inducing membrane damage of varying intensities, nodal Nav1.6 channels should immediately "leak" over a broadly left-smeared window current range.

A model for studying the energetics of sustained high frequency firing.

Regulating membrane potential and synaptic function contributes significantly to the energetic costs of brain signaling, but the relative costs of action potentials (APs) and synaptic transmission during high-frequency firing are unknown. The continuous high-frequency (200-600Hz) electric organ discharge (EOD) of Eigenmannia, a weakly electric fish, underlies its electrosensing and communication. EODs reflect APs fired by the muscle-derived electrocytes of the electric organ (EO). Cholinergic synapses at the excitable posterior membranes of the elongated electrocytes control AP frequency. Based on whole-fish O2 consumption, ATP demand per EOD-linked AP increases exponentially with AP frequency. Continual EOD-AP generation implies first, that ion homeostatic processes reliably counteract any dissipation of posterior membrane ENa and EK and second that high frequency synaptic activation is reliably supported. Both of these processes require energy. To facilitate an exploration of the expected energy demands of each, we modify a previous excitability model and include synaptic currents able to drive APs at frequencies as high as 600 Hz. Synaptic stimuli are modeled as pulsatile cation conductance changes, with or without a small (sustained) background conductance. Over the full species range of EOD frequencies (200-600 Hz) we calculate frequency-dependent "Na+-entry budgets" for an electrocyte AP as a surrogate for required 3Na+/2K+-ATPase activity. We find that the cost per AP of maintaining constant-amplitude APs increases nonlinearly with frequency, whereas the cost per AP for synaptic input current is essentially constant. This predicts that Na+ channel density should correlate positively with EOD frequency, whereas AChR density should be the same across fish. Importantly, calculated costs (inferred from Na+-entry through Nav and ACh channels) for electrocyte APs as frequencies rise are much less than expected from published whole-fish EOD-linked O2 consumption. For APs at increasingly high frequencies, we suggest that EOD-related costs external to electrocytes (including packaging of synaptic transmitter) substantially exceed the direct cost of electrocyte ion homeostasis.

Dual stretch responses of mHCN2 pacemaker channels: accelerated activation, accelerated deactivation.

Mechanoelectric feedback in heart and smooth muscle is thought to depend on diverse channels that afford myocytes a mechanosensitive cation conductance. Voltage-gated channels (e.g., Kv1) are stretch sensitive, but the only voltage-gated channels that are cation permeant, the pacemaker or HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, have not been tested. To assess if HCN channels could contribute to a mechanosensitive cation conductance, we recorded I(HCN) in cell-attached oocyte patches before, during, and after stretch for a range of voltage protocols. I(mHCN2) has voltage-dependent and instantaneous components; only the former was stretch sensitive. Stretch reversibly accelerated hyperpolarization-induced I(mHCN2) activation (likewise for I(spHCN)) and depolarization-induced deactivation. HCN channels (like Kv1 channels) undergo mode-switch transitions that render their activation midpoints voltage history dependent. The result, as seen from sawtooth clamp, is a pronounced hysteresis. During sawtooth clamp, stretch increased current magnitudes and altered the hysteresis pattern consistent with stretch-accelerated activation and deactivation. I(mHCN2) responses to step protocols indicated that at least two transitions were mechanosensitive: an unspecified rate-limiting transition along the hyperpolarization-driven path, mode I(closed)-->mode II(open), and depolarization-induced deactivation (from mode I(open) and/or from mode II(open)). How might this affect cardiac rhythmicity? Since hysteresis patterns and "on" and "off" I(HCN) responses all changed with stretch, predictions are difficult. For an empirical overview, we therefore clamped patches to cyclic action potential waveforms. During the diastolic potential of sinoatrial node cell and Purkinje fiber waveforms, net stretch effects were frequency dependent. Stretch-inhibited (SI) I(mHCN2) dominated at low frequencies and stretch-augmented (SA) I(mHCN2) was progressively more important as frequency increased. HCN channels might therefore contribute to either SI or SA cation conductances that in turn contribute to stretch arrhythmias and other mechanoelectric feedback phenomena.

Membrane stretch slows the concerted step prior to opening in a Kv channel.

In the simplest model of channel mechanosensitivity, expanded states are favored by stretch. We showed previously that stretch accelerates voltage-dependent activation and slow inactivation in a Kv channel, but whether these transitions involve expansions is unknown. Thus, while voltage-gated channels are mechanosensitive, it is not clear whether the simplest model applies. For Kv pore opening steps, however, there is excellent evidence for concerted expansion motions. To ask how these motions respond to stretch, therefore, we have used a Kv1 mutant, Shaker ILT, in which the step immediately prior to opening is rate limiting for voltage-dependent current. Macroscopic currents were measured in oocyte patches before, during, and after stretch. Invariably, and directly counter to prediction for expansion-derived free energy, ILT current activation (which is limited by the concerted step prior to pore opening) slowed with stretch and the g(V) curve reversibly right shifted. In WTIR (wild type, inactivation removed), the g(V) (which reflects independent voltage sensor motions) is left shifted. Stretch-induced slowing of ILT activation was fully accounted for by a decreased basic forward rate, with no change of gating charge. We suggest that for the highly cooperative motions of ILT activation, stretch-induced disordering of the lipid channel interface may yield an entropy increase that dominates over any stretch facilitation of expanded states. Since tail current tau(V) reports on the opposite (closing) motions, ILT and WTIR tau(V)(tail) were determined, but the stretch responses were too complex to shed much light. Shaw is the Kv3 whose voltage sensor, introduced into Shaker, forms the chimera that ILT mimics. Since Shaw2 F335A activation was reportedly a first-order concerted transition, we thought its activation might, like ILT's, slow with stretch. However, Shaw2 F335A activation proved to be sigmoid shaped, so its rate-limiting transition was not a concerted pore-opening transition. Moreover, stretch, via an unidentified non-rate-limiting transition, augmented steady-state current in Shaw2 F335A. Since putative area expansion and compaction during ILT pore opening and closing were not the energetically consequential determinants of stretch modulation, models incorporating fine details of bilayer structural forces will probably be needed to explain how, for Kv channels, bilayer stretch slows some transitions while accelerating others.

Lipid stress at play: mechanosensitivity of voltage-gated channels.

Membrane stretch modulates the activity of voltage-gated channels (VGCs). These channels are nearly ubiquitous among eukaryotes and they are present, too, in prokaryotes, so the potential ramifications of VGC mechanosensitivity are diverse. In situ traumatic stretch can irreversibly alter VGC activity with lethal results but that is pathology. This chapter discusses the reversible responses of VGCs to stretch, with the general relation of stretch stimuli to other forms of lipid stress, and briefly, with some irreversible stretch effects (=stretch trauma). A working assumption throughout is that mechanosensitive (MS) VGC motions-that is, motions that respond reversibly to bilayer stretch-are susceptible to other forms of lipid stress, such as the stresses produced when amphiphilic molecules (anesthetics, lipids, alcohols, and lipophilic drugs) are inserted into the bilayer. Insofar as these molecules change the bilayer's lateral pressure profile, they can be termed bilayer mechanical reagents (BMRs). The chapter also discusses the MS VGC behavior against the backdrop of eukaryotic channels more widely accepted as "MS channels"--namely, the transient receptor potential (TRP)-based MS cation channels.

Studying the mechanosensitivity of voltage-gated channels using oocyte patches.

The mechanosensitivity of voltage-gated (VG) channels is of biophysical, physiological. and pathophysiological interest. Xenopus oocytes offer a critical advantage for investigating the electrophysiology of recombinant VG channels subjected to membrane stretch, namely, the ability to monitor macroscopic current from membrane patches. High-density channel expression in oocytes makes for macroscopic current in conventional-size, mechanically sturdy patches. With the patch configuration, precisely the same membrane that is voltage-clamped is the membrane subjected to on-off stretch stimuli. With patches, meaningful stretch dose responses are possible. Experimental design should facilitate within-patch comparisons wherever possible. The mechanoresponses of some VG channels depend critically on patch history. Methods for minimizing and coping with interference from endogenous voltage-dependent and stretch-activated endogenous channels are described.

The Hv1 proton channel responds to mechanical stimuli.

The voltage-gated proton channel, Hv1, is expressed in tissues throughout the body and plays important roles in pH homeostasis and regulation of NADPH oxidase. Hv1 operates in membrane compartments that experience strong mechanical forces under physiological or pathological conditions. In microglia, for example, Hv1 activity is potentiated by cell swelling and causes an increase in brain damage after stroke. The channel complex consists of two proton-permeable voltage-sensing domains (VSDs) linked by a cytoplasmic coiled-coil domain. Here, we report that these VSDs directly respond to mechanical stimuli. We find that membrane stretch facilitates Hv1 channel opening by increasing the rate of activation and shifting the steady-state activation curve to less depolarized potentials. In the presence of a transmembrane pH gradient, membrane stretch alone opens the channel without the need for strong depolarizations. The effect of membrane stretch persists for several minutes after the mechanical stimulus is turned off, suggesting that the channel switches to a "facilitated" mode in which opening occurs more readily and then slowly reverts to the normal mode observed in the absence of membrane stretch. Conductance simulations with a six-state model recapitulate all the features of the channel's response to mechanical stimulation. Hv1 mechanosensitivity thus provides a mechanistic link between channel activation in microglia and brain damage after stroke.

Membrane tension accelerates rate-limiting voltage-dependent activation and slow inactivation steps in a Shaker channel.

A classical voltage-sensitive channel is tension sensitive--the kinetics of Shaker and S3-S4 linker deletion mutants change with membrane stretch (Tabarean, I.V., and C.E. Morris. 2002. Biophys. J. 82:2982-2994.). Does stretch distort the channel protein, producing novel channel states, or, more interestingly, are existing transitions inherently tension sensitive? We examined stretch and voltage dependence of mutant 5aa, whose ultra-simple activation (Gonzalez, C., E. Rosenman, F. Bezanilla, O. Alvarez, and R. Latorre. 2000. J. Gen. Physiol. 115:193-208.) and temporally matched activation and slow inactivation were ideal for these studies. We focused on macroscopic patch current parameters related to elementary channel transitions: maximum slope and delay of current rise, and time constant of current decline. Stretch altered the magnitude of these parameters, but not, or minimally, their voltage dependence. Maximum slope and delay versus voltage with and without stretch as well as current rising phases were well described by expressions derived for an irreversible four-step activation model, indicating there is no separate stretch-activated opening pathway. This model, with slow inactivation added, explains most of our data. From this we infer that the voltage-dependent activation path is inherently stretch sensitive. Simulated currents for schemes with additional activation steps were compared against datasets; this showed that generally, additional complexity was not called for. Because the voltage sensitivities of activation and inactivation differ, it was not possible to substitute depolarization for stretch so as to produce the same overall PO time course. What we found, however, was that at a given voltage, stretch-accelerated current rise and decline almost identically--normalized current traces with and without stretch could be matched by a rescaling of time. Rate-limitation of the current falling phase by activation was ruled out. We hypothesize, therefore, that stretch-induced bilayer decompression facilitates an in-plane expansion of the protein in both activation and inactivation. Dynamic structural models of this class of channels will need to take into account the inherent mechanosensitivity of voltage-dependent gating.

Mechanosensitive gating of Kv channels.

K-selective voltage-gated channels (Kv) are multi-conformation bilayer-embedded proteins whose mechanosensitive (MS) Popen(V) implies that at least one conformational transition requires the restructuring of the channel-bilayer interface. Unlike Morris and colleagues, who attributed MS-Kv responses to a cooperative V-dependent closed-closed expansion↔compaction transition near the open state, Mackinnon and colleagues invoke expansion during a V-independent closed↔open transition. With increasing membrane tension, they suggest, the closed↔open equilibrium constant, L, can increase >100-fold, thereby taking steady-state Popen from 0→1; "exquisite sensitivity to small…mechanical perturbations", they state, makes a Kv "as much a mechanosensitive…as…a voltage-dependent channel". Devised to explain successive gK(V) curves in excised patches where tension spontaneously increased until lysis, their L-based model falters in part because of an overlooked IK feature; with recovery from slow inactivation factored in, their g(V) datasets are fully explained by the earlier model (a MS V-dependent closed-closed transition, invariant L≥4). An L-based MS-Kv predicts neither known Kv time courses nor the distinctive MS responses of Kv-ILT. It predicts Kv densities (hence gating charge per V-sensor) several-fold different from established values. If opening depended on elevated tension (L-based model), standard gK(V) operation would be compromised by animal cells' membrane flaccidity. A MS V-dependent transition is, by contrast, unproblematic on all counts. Since these issues bear directly on recent findings that mechanically-modulated Kv channels subtly tune pain-related excitability in peripheral mechanoreceptor neurons we undertook excitability modeling (evoked action potentials). Kvs with MS V-dependent closed-closed transitions produce nuanced mechanically-modulated excitability whereas an L-based MS-Kv yields extreme, possibly excessive (physiologically-speaking) inhibition.

Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels: (embryogenesis/mechanosensitive ion channels/channel blockers/Xenopus/ascidians).

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch-activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd3+ ) concentrations up to 100 times the Kd for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd3+ , tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.