The Kinetics and the Permeation Properties of Piezo Channels.

Piezo channels are eukaryotic, cation-selective mechanosensitive channels (MSCs), which show rapid activation and voltage-dependent inactivation. The kinetics of these channels are largely consistent across multiple cell types and different stimulation paradigms with some minor variability. No accessory subunits that associate with Piezo channels have been reported. They are homotrimers and each ∼300kD monomer has an N-terminal propeller blade-like mechanosensing module, which can confer mechanosensing capabilities on ASIC-1 (the trimeric non-MSC, acid-sensing ion channel-1) and a C-terminal pore module, which influences conductance, selectivity, and channel inactivation. Repeated stimulation can cause domain fracture and diffusion of these channels leading to synchronous loss of inactivation. The reconstituted channels spontaneously open only in asymmetric bilayers but lack inactivation. Mutations that cause hereditary xerocytosis alter PIEZO1 kinetics. The kinetics of the wild-type PIEZO1 and alterations thereof in mutants (M2225R, R2456K, and DhPIEZO1) are summarized in the form of a quantitative model and hosted online. The pore is permeable to alkali ions although Li+ permeates poorly. Divalent cations, notably Ca2+, traverse the channel and inhibit the flux of monovalents. The large monovalent organic cations such as tetramethyl ammonium and tetraethyl ammonium can traverse the channel, but slowly, suggesting a pore diameter of ∼8Å, and the estimated in-plane area change upon opening is around 6-20nm2. Ruthenium red can enter the channel only from the extracellular side and seems to bind in a pocket close to residue 2496.

Free Volume in Membranes: Viscosity or Tension?

Many papers have used fluorescent probe diffusion to infer membrane viscosity but the measurement is actually an assay of the free volume of the membrane. The free volume is also related to the membrane tension. Thus, changes in probe mobility refer equally well to changes in membrane tension. In complicated structures like cell membranes, it appears more intuitive to consider variations in free volume as referring to the effect of domains structures and interactions with the cytoskeleton than changes in viscosity since tension is a state variable and viscosity is not.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Membrane Electromechanics in Biology, with a Focus on Hearing.

Cells are ion conductive gels surrounded by a ~5-nm-thick insulating membrane, and molecular ionic pumps in the membrane establish an internal potential of approximately -90 mV. This electrical energy store is used for high-speed communication in nerve and muscle and other cells. Nature also has used this electric field for high-speed motor activity, most notably in the ear, where transduction and detection can function as high as 120 kHz. In the ear, there are two sets of sensory cells: the "inner hair cells" that generate an electrical output to the nervous system and the more numerous "outer hair cells" that use electromotility to counteract viscosity and thus sharpen resonance to improve frequency resolution. Nature, in a remarkable exhibition of nanomechanics, has made out of soft, aqueous materials a microphone and high-speed decoder capable of functioning at 120 kHz, limited only by thermal noise. Both physics and biology are only now becoming aware of the material properties of biomembranes and their ability to perform work and sense the environment. We anticipate new examples of this biopiezoelectricity will be forthcoming.

Thermodynamics of mechanosensitivity.

Mechanosensitivity of ion channels is conventionally interpreted as being driven by a change of their in-plane cross-sectional area A(msc). This, however, does not include any factors relating to membrane stiffness, thickness, spontaneous curvature or changes in channel shape, length or stiffness. Because the open probability of a channel is sensitive to all these factors, we constructed a general thermodynamic formalism. These equations provide the basis for the analysis of the behaviour of mechanosensitive channels in lipids of different geometric and chemical properties such as the hydrophobic mismatch at the boundary between the protein and lipid or the effects of changes in the bilayer intrinsic curvature caused by the adsorption of amphipaths. This model predicts that the midpoint gamma(1/2) and the slope(1/2) of the gating curve are generally not independent. Using this relationship, we have predicted the line tension at the channel/lipid border of MscL as approximately 10 pN, and found it to be much less than the line tension of aqueous pores in pure lipid membranes. The MscL channel appears quite well matched to its lipid environment. Using gramicidin as a model system, we have explained its observed conversion from stretch-activated to stretch-inactivated gating as a function of bilayer thickness and composition.

Adapting the Quesant Nomad atomic force microscope for biology and patch-clamp atomic force microscopy.

The Quesant Nomad atomic force microscope (AFM) was modified to produce a reliable patch-clamp AFM for demanding biologic applications. The AFM's laser optics forms the basis of a condenser that allows simultaneous Köhler illumination and AFM imaging on an inverted optical microscope. The original AFM scan head was replaced with plastic and glass to make it biologically inert. A bevel cut in the new scan head permits clearance for patch clamp pipets. Cantilevers are attached to the scan head with a quick setting silicone rubber that is readily removable. Software was developed to (a) automate a gentle approach and set a specific feedback force, (b) provide a mouse-driven control of the X-Y position of the probe tip and recall of saved locations, and (c) measure force-distance curves over user defined paths. Additional modifications were made to minimize mechanical noise. The patch-clamp AFM achieves 600 fA (3 kHz bandwidth) and 1 A RMS noise levels (10 kHz bandwidth). The correlation of electrical and mechanical information allows signal averaging and measures sub-Angstrom, sub-millisecond electromotile responses from cells.

Voltage-induced membrane movement.

Thermodynamics predicts that transmembrane voltage modulates membrane tension and that this will cause movement. The magnitude and polarity of movement is governed by cell stiffness and surface potentials. Here we confirm these predictions using the atomic force microscope to dynamically follow the movement of voltage-clamped HEK293 cells in different ionic-strength solutions. In normal saline, depolarization caused an outward movement, and at low ionic strength an inward movement. The amplitude was proportional to voltage (about 1 nm per 100 mV) and increased with indentation depth. A simple physical model of the membrane and tip provided an estimate of the external and internal surface charge densities (-5 x 10(-3) C x m(-2) and -18 x 10(-3) C x m(-2), respectively). Salicylate (a negative amphiphile) inhibited electromotility by increasing the external charge density by -15 x 10(-3) C x m(-2). As salicylate blocks electromotility in cochlear outer hair cells at the same concentration, the role of prestin as a motor protein may need to be reassessed.

Stretch-induced endothelin-1 production by astrocytes.

Astrocytes proliferate during central nervous system (CNS) development and then remain quiescent. However, at a site of brain injury, astrocytes re-enter the cell cycle and undergo complex biochemical/functional changes known as reactive gliosis. Gliosis is the most important histopathologic indicator of CNS injury, regardless of etiology. Endothelins (ETs) have powerful mitogenic effects on astrocytes and have recently been implicated in the induction of gliosis. Reactive astrocytes produce, store. secrete and bind endothelin-1 (ET-1). The stimuli responsible for activating ET production in astrocytes are unresolved. Because of the relationship between stretch and ET production in other cell types, and the observation that ET-1-positive reactive astrocytes appear in mechanically deformed regions, we are examining whether mechanical deformation affects ET-1 production. We expose mature rat astrocyte cultures to mechanical stress using flexible-bottomed culture plates. Mechanical stretch of quiescent, confluent cultures causes an increase in cytoplasmic Ca2+ and inositol trisphosphate (IP3), and a substantial increase in ET-1 production and secretion into the culture media.

A direct optimization approach to hidden Markov modeling for single channel kinetics.

Hidden Markov modeling (HMM) provides an effective approach for modeling single channel kinetics. Standard HMM is based on Baum's reestimation. As applied to single channel currents, the algorithm has the inability to optimize the rate constants directly. We present here an alternative approach by considering the problem as a general optimization problem. The quasi-Newton method is used for searching the likelihood surface. The analytical derivatives of the likelihood function are derived, thereby maximizing the efficiency of the optimization. Because the rate constants are optimized directly, the approach has advantages such as the allowance for model constraints and the ability to simultaneously fit multiple data sets obtained at different experimental conditions. Numerical examples are presented to illustrate the performance of the algorithm. Comparisons with Baum's reestimation suggest that the approach has a superior convergence speed when the likelihood surface is poorly defined due to, for example, a low signal-to-noise ratio or the aggregation of multiple states having identical conductances.

Hidden Markov modeling for single channel kinetics with filtering and correlated noise.

Hidden Markov modeling (HMM) can be applied to extract single channel kinetics at signal-to-noise ratios that are too low for conventional analysis. There are two general HMM approaches: traditional Baum's reestimation and direct optimization. The optimization approach has the advantage that it optimizes the rate constants directly. This allows setting constraints on the rate constants, fitting multiple data sets across different experimental conditions, and handling nonstationary channels where the starting probability of the channel depends on the unknown kinetics. We present here an extension of this approach that addresses the additional issues of low-pass filtering and correlated noise. The filtering is modeled using a finite impulse response (FIR) filter applied to the underlying signal, and the noise correlation is accounted for using an autoregressive (AR) process. In addition to correlated background noise, the algorithm allows for excess open channel noise that can be white or correlated. To maximize the efficiency of the algorithm, we derive the analytical derivatives of the likelihood function with respect to all unknown model parameters. The search of the likelihood space is performed using a variable metric method. Extension of the algorithm to data containing multiple channels is described. Examples are presented that demonstrate the applicability and effectiveness of the algorithm. Practical issues such as the selection of appropriate noise AR orders are also discussed through examples.

Mechanically induced calcium movements in astrocytes, bovine aortic endothelial cells and C6 glioma cells.

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca(2+). It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca(2+) is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca(2+). In these three cell types, no influx of ions is required for Ca(2+) elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca(2+). The Ca(2+) response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP(3)-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn(2+) influx pathway. This Mn(2+) pathway may be mechanosensitive channels, Ca(2+)-activated cation channels or depletion-activated Ca(2+) channels.

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

Activation and inactivation of mechanosensitive currents in the chick heart.

The behavior of MS channels in embryonic chick ventricular myocytes activated by direct mechanical stimulation is strongly affected by inactivation. The amplitude of the current is dependent not only on the amplitude of the stimulus, but also the history of stimulation. The MS current inactivation appears to be composed of at least two contributions: (i) rearrangement of the cortical tension transducing elements and (ii) blocking action of an autocrine agent released from the cell. With discrete mechanical stimuli, the MS current amplitude in the second press of a double press protocol was always smaller than the amplitude of the first MS current. Occasionally, a large MS current occurred when the cell was first stimulated, but subsequently the cell became unresponsive. For a series of stimuli of varying amplitudes, the order in which they were applied to the cell affected the size of the observed MS current for a given stimulus magnitude. When continuous sinusoidal stimulation was applied to the cells, the MS current envelope either reached a steady state, or inactivated. With sinusoidal stimulation, the MS response could be enhanced or restored by simple perfusion of fluid across the cell. This suggests that mechanical stimulation of the cells produces an autocrine inhibitor of MS channels as well as resulting in cortical rearrangement.

Whole-cell mechanosensitive currents in rat ventricular myocytes activated by direct stimulation.

Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive. Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation. The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.

Stretch-activated whole cell currents in adult rat cardiac myocytes.

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 microM Gd(3+) but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately -6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 - 5.0 SL (microm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

Inactivation of P2X2 purinoceptors by divalent cations.

1. P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2. The ability to inactivate whole-cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (approximately 100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3. The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co-operativity with ionic species suggests the presence of experimentally unresolved ligand-insensitive kinetic steps. 4. Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+-binding sites are probably located near the extracellular surface of the membrane. 5. The recovery from inactivation was slow, with a time constant of approximately 7 min. 6. Ca2+-sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7. Inactivation rates observed in different patch configurations suggest that the induction of Ca2+-dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.

Ion permeation and block of P2X(2) purinoceptors: single channel recordings.

P2X(2) purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG(+), Tris(+), TMA(+), and TEA(+). The selectivity sequence for currents carried by alkali metal ions is: K(+) > Rb(+) > Cs(+) > Na(+) > Li(+), which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG(+), Tris(+), TMA(+) and TEA(+) were virtually impermeant. The divalent ions (Mn(2+), Mg(2+), Ca(2+) and Ba(2+)) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn(2+) > Mg(2+) > Ca(2+) > Ba(2+).

Practical limits on the maximal speed of solution exchange for patch clamp experiments.

Studying ligand-gated ion channels often requires the ability to change solutions quickly. Using finite element models, I have examined the practical limitations of how fast solutions can be exchanged on an outside-out patch using a dual stream switcher. The primary factors controlling the speed of response are the flow velocity, proximity of the patch to the exit ports, the width of the partition between the two streams, the velocity with which the streams can be moved across the patch, and the viscosity of the solutions. The practical limit seems to be a rise time of approximately 20 microseconds. The rate-limiting step is the velocity of the (usually piezo) motor that translates the streams across the patch. Increasing the perfusate viscosity improves speed by slowing dissipation of the concentration gradients. A flow switcher can also be used for bipolar temperature jumps with a rise time of approximately 100 microseconds.