Phylogenetic relationships among DEG/ENaC proteins. Nematode degenerins are shown with blue lines. The current degenerin content of the complete nematode genome is included. The seven genetically characterized (DEG-1, DEL-1, FLR-1, MEC-4, MEC-10, UNC-8 and UNC-105) are shown in red. Representative DEG/ENaC proteins from a variety of organisms, ranging from snails to humans, are also included (mammalian: red lines; fly: green lines; snail: orange line). The scale bar denotes relative evolutionary distance equal to 0.1 nucleotide substitutions per site [119].

Punctuate localization of a putative mechanosensitive ion channel subunit. Image of an AVM touch receptor neuron expressing a GFP-tagged MEC-4 protein. Fluorescence is unevenly distributed along the process of the neuron in distinct puncta, which may represent the location of the mechanotransducing apparatus.

Schematic representation and topology of the MEC-2 protein. Conserved domains as well as hydrophobic regions are highlighted. Putative interactions with the degenerin channel and the cytoskeleton are indicated [54].

Phylogenetic relations among SPFH domain proteins. A dendrogram showing distance relationships among most of the stomatin protein super-family members (the complete ClustalW generated alignment on which the dendrogram was based is available at http://www.imbb.forth.gr/worms/worms/alignment.gif). The dendrogram was constucted with the neighbor-joining method (120) based on pairwise distance estimates of the expected number of amino acid replacements per site (0.10 in the scale bar), and visualized by TreeTool (http,//geta.life.uiuc.edu). Protein sub-families are denoted in different colors [136].

A model for UNC-8 involvement in stretch-regulated control of locomotion. Schematic diagram of potentiated and inactive VB class motor neurons. Neuro-muscular junctions (signified by triangles) are made near the cell body [135, 151]. Mechanically-activated channels postulated to include UNC-8 (and, possibly in VB motor neurons, DEL-1) subunits (signified by Y figures) are hypothesized to be concentrated at the synapse-free, undifferentiated ends of the VB neuron. Mechanically-gated channels could potentiate local excitation of muscle. Body stretch is postulated to activate mechanically-gated channels which potentiate the motor neuron signal that excites a specific muscle field. Sequential activation of motor neurons that are distributed along the ventral nerve cord and signal non-overlapping groups of muscles, amplifies and propagates the sinusoidal body wave (NMJ: neuromuscular junction).

A mechanotransducing complex in C. elegans touch receptor neurons. In the absence of mechanical stimulation the channel is closed and therefore the sensory neuron is idle. Application of a mechanical force to the body of the animal results in distortion of a network of interacting molecules that opens the degenerin channel. Na⁺ influx depolarizes the neuron initiating the preceptory integration of the stimulus [47].

Broad interactions involving Mechanically Mediated Crosstalk (MMC). Crosstalk is Mediated between membrane channels 1 & 2 (e.g. (Northwest and Northeast) stretch activated channels (SAC), and ATP activated potassium (KATP) channel), as well as receptors 1 and 2 (e.g. (equator) (β receptor & angiotensin receptor (AT)). MMC acting at membrane and cell signal level can also globally affect the heart, influencing physiology, and clinico pathology (Southwest and Southeast). Cytoskeletal and cell signal changes are invoked during MMC.

Conjectural diagram of Mechanically Mediated Crosstalk (MMC) at cellular level. "Stress strain" represents intra and extracellular mechanical input - through extracellular matrix as well, e.g. physiological and biophysical force and pressure changes, including pathophysiological ones e.g. dilated cardiomyopathy, dyskinesia These stress/strains impinge on stretch activated channels (SAC at 12 o'clock), which open to admit charge-carrying ions such as Ca - downward arrow, and Na - curved arrow. Stress/strains activate, diagrammatically, all the moieties on the figurative cell membrane, transmitted via extracellular matrix, integral & associated focal adhesion kinase (FAK), and the cytoskeleton (designated at 6 o'clock). This stress/strain transmission is indicated clockwise from SAC:- at 1 o'clock - Ion exchangers Na/Ca (electrogenic) & Na/H. Intracellular Na rise will increase intracellular Ca. Further round, at 3 o'clock translocation (TRANS) of phospholipase C dependent kinase (PKC) to the membrane, embracing 1 ;2 diacylglycerol (DAG) and the (particular) receptor activated C kinase (RACK) bringing it near its designated protein. In this way, hypertrophy, via mitogen activated phosphokinase (MAPK) with tyrosine kinase, and immediate early genes (IEG), encompass PKC. Its translocation to the membrane may also be a contributory mechanism in preconditioning: at 4 o'clock - Phospholipase C (PLC), via phosphotidylinositol (Pi) produces inositol trisphosphate (IP3), and also cleaves the phosphorylated base forming DAG: the former activating Ca2⁺, and the latter PKC: at 5 o'clock -AT angiotensin, ET endothelin via Gq protein impinges on PLC: at 6 o'clock integrin & associated focal adhesion kinase (FAK) connect the cytoskeleton with extracellular matrix: at 7 o'clock - L type calcium channel (L_ca: at 8 o'clock 2 pore (tandem pore) forming K channels (2-PK) - still controversial as to the extent they exist in myocardium: at 9 o'clock - ATP activated potassium (K_ATP) channel: at 11 o'clock "autonomic" receptors. Acetyl choline (M2) and (β? receptors with their respective G proteins Gi & Gs. Here mechanosensitivity of second messengers seem likely, including parts of the cascade in which adenyl cyclase (Ad Cy) splits ATP giving cyclic adenosine monophosphate (cAMP), involving G protein, to affect the LCa channel via phosphokinase A (PKA). Aditionally, ATP can activate the potassium channels, K_ATP and 2-PK.

Net membrane currents modulated by local axial stretch. A: Currents in response to 120 ms clamp steps that depolarize from -45 mV (holding potential) for 70 ms to either +10 mV or to 80 mV. Back trace at bottom indicates the pulse protocol. Black tracing before, red one during 10 μm local axial stretch. Mouse ventricular myocyte, unpublished. B: stretch sensitive current as difference of the red minus black current trace from panel A. Note: the stretch induced current is negative at -80 and -45 mV, however, positive at +10 mV. C: iv curve measured with a ramp voltage command repolarizing the membrane from +60 mV to 100 mV within 1.6s. Blue record before, red record during 10 μm local axial stretch.

Mechanosensitivity of K⁺ currents. A: Iv curves of net membrane currents measured with ramp commands repolarizing the cell from +60 to -100 mV at a rate of 100 mV/s. Blue trace before, red trace during 10 μm stretch. Arrows mark the shift of resting potential from -90 to -70 mV, the reduction of the hump of outward current at -60 mV and the increase in outward current at +50 mV. B: The N-shape of the control i-v curve is attributed to the inward rectifier I_K1, the driving force (V_m E_K is multiplied with a conductance G_K1 that deactivates when the membrane is depolarized (blue curve). C: Modelling the control currents by superimposition of I_K1 with an outwardly rectifying K⁺ current 1_K0. D: Modelling the currents during stretch. In addition to the activation of I_SAC, deactivation of I_K1 is necessary to model reduced the reduced slope and the reduced hump of outward current (-60mV) of the iv curve. In addition, the increase in outward current at positive potentials needs the postulate that stretch enhances I_K0. (Unpublished).

Stretch-induced increments in free cytoplasmic Na⁺ concentration [Na⁺]_c analyzed by Sodium Green fluorescence in a camera imaging system. Guinea pig ventricular myocyte stimulated at 2 Hz. A: Image before stretch, position of the glass stylus (S) and of the patch pipette (PP) indicated. B: Image 2 min after start of 10 μm stretch. C: Image 3 min after start of 10 μm stretch. Note: The appearance of red and white in the pseudocolor code indicates an 2-fold or 3-fold increase in [Na⁺]_c. (From Isenberg et al. [32], with permission).

Stretch-induced changes in the cytosolic Ca²⁺ concentration [Ca²⁺]_c as analyzed pseudoratiometrically in the confocal microscope. Mouse ventricular myocyte loaded by 5 μM Fluo-4AM, depolarized from -45 to 0 mV for 40 ms at 1 Hz. Upper panels: Scan lines from top to bottom before (left) and during 10 μm stretch (right). Darkness of pixels increases with higher [Ca²]_c. Aa before, Ba during 10 μrn local stretch. Dotted line labels upper cell edge before stretch that is displaced by 5 μm during 10 μm local stretch (Ba). Arrows mark the occurence of sparks during stretch. Lower panels: Ca²⁺ transients, averaged from the linescans. During stretch (Bb), the peak (marked by circle) is 10% larger and the decay is faster (half time marked by circle). Inset: Contraction (edge movement) is faster during stretch (blue trace). Murine ventricular myocytes, unpublished.

Analysis of stretch induced changes in the total concentration (σ) of sodium (σNa) and calcium (σCa) with electronprobe microanalysis (EPMA). A: longitudinal cryosection of murine ventricular myocytes frozen after stimulation without stretch (left panel) and after stimulation and 2 min stretch (right panel). Nucleus (n), nuclear envelope (nu), mitochondria (m), z-lines (z) are well recognizable. B: σ[Na]-map. The elemental spectra were collected pixel by pixel (pixel size 16 nm, 40 s dwell time) in the region included in the orange frame of the cyrosection from the control myocytes (left panel) and form the stretched myocytes (right panel). σ[Na] are given in full false colors. N: nucleus, cy: cytosol. C: Upper maps. σ[Ca] in full false color scale in the mapped regions of the two cells. Left panel = control cell, right panel =stretched cell. Lower maps: red pixel with σ[Ca] higher than 8 mmol (kg dw)^-1 were superimposed to the simultaneously collected phosphor map (grey image) in order to better attribute the concentrations of the "hot spots" if σ[Ca] to membrane structures. Whereas in the control cell only few pixel with high [Ca] are detectable in the nuclear envelope and in the nucleus (most "hot spots" are on perinuclear organelles), in the stretched cell a large number of pixel with high σ[Ca] are shown in nuclear envelope as well as just internal (nucleus) and external (cytosol) to it. The maps or the two experiments were collected under identical analysis conditions. Elemental digital maps confirm the data of static analysis. (From Kondratev and Gallitelli [44], with permission).

The model of biatrial dilatation in the Langendorff-perfused rabbit heart Left - Schematic diagram of the method to vary the atrial pressure. All caval and pulmonary veins were ligated and the perfusion fluid flowing out of the coronary sinus could leave the heart exclusively through a cannula in the pulmonary artery (PA). The interatrial septum was perforated and a Y-shaped manometer was inserted both into the superior caval vein (SVC) and one of the pulmonary veins (PV). In this preparation the biatrial pressure could be varied simply by adjusting the height of the pulmonary outflow cannula. A monophasic action potential (MAP) catheter was introduced into the right atrial cavity through the inferior caval vein (IVC) to record atrial MAPs from the right atrial mid-wall. Right - Photographs of the right atrium at an atrial pressure of 0 cm H₂O (top) and 10 cm H₂O (bottom) (modified from Ravelli and Allessie [26], with permission).

Fura-2 measurements of [Ca²⁺]_i (red trace) in a leech AP neuron at rest, as it swells in hypotonic solution and as it reshrinks. Normalized values of membrane area are plotted (blue trace) Representative ratio images (340/380 nm) at the times marked a, b and c are displayed.

Measurements of [Ca²⁺]_i (red trace) and membrane area (blue trace) at rest, during hypotonic swelling in the presence of 10 M Gd⁺³ (+bl) and after removal of the blocker (-bl). Representative ratio images at the times marked a, b and c are displayed.

Image (A) and schematic diagram (B) of the pressure step system. EV1 and EV2: solenoid valves; T: piezoresistive pressure transducer; H: pipette holder; D: tap; M: microchamber. A SAC response (upper trace) to a pulse of 50 mmHg (lower trace), at a steady membrane potential of 50 mV, is illustrated in C. EV1 opens between a and b, while EV2 opens between c and d. The inside-out membrane patch was excised from an AP cell body and contained two channels.

Model of signal transduction pathways in mechanotransduction. Abbreviations: EP2 prostaglandin receptor 2, 7TR seven transmembrane helix receptor, G GTP-binding Protein, cAMP cyclic adenosine monophoshate, PKA protein kinase A, PKC protein kinase C, MAP Kinase mitogen-activated kinase, TRE tetradecanoylphorbol acetate-response element, CRE cAMP-response element, iNOS inducible nitric oxide synthase, COX-2 cycloxygenase-2.

Immunohistochemical localization of the subunit of ENaC in tissues represented on human tissue microarrays (TMAs) obtained from the Cooperative Human Tissue Network (CHTN) of the National Cancer Institute (NCI), the National Institutes of Health, Bethesda, MD ( http://facu1ty.virginia.edu/chtn-tma/home.html ). ?ENaC expression is shown in the human renal cortex (A), renal medulla (B), urinary bladder (C) and articular cartilage (D). ENaC expression is particularly abundant in principal cells of the distal nephron (cortical and medullary collecting ducts).

Expression of the and subunits of ENaC in C20/A4 human chondrocyte-like cells. In the cell line the and subunits appear to be localized in different subcellular compartments: immunostaining for the subunit is seen in the plasma membrane whereas the subunit is predominantly intracellular. Bars represent 10 μm (approx.).