Impedance of a Small Electrically Isolated Area of the Muscle Cell Surface

A method has been developed permitting measurement of membrane impedance and current, as a function of transmembrane potential, at small, electrically isolated regions of the muscle cell surface without microelectrode impalement. The frequency dependence of the muscle cell membrane capacity found earlier by other methods has been confirmed. The average capacity is 3.5 µf/cm² with a phase angle of 71° at 5 kilocycles. The internal phase angle of the complex impedance plot of whole muscle probably does not result from a distribution of fiber diameters and membrane capacities, since it also appears in the present experiments where measurements are confined to a small region of a single fiber.     

The wave of activation current in the egg of the medaka fish

An extracellular vibrating electrode was used to measure the ring-shaped wave of inward current, the activation current, that propagates at 10 micron/sec across the egg of the medaka fish, Oryzias latipes, from the site of sperm-egg fusion at the animal pole to the vegetal pole. This activation wave is due to a localized increase in the conductance to Na+, K+, and Ca2+ and reflects the propagated opening of these ion channels. The earliest detectable current begins to enter the animal pole 20 sec after the initiation of the fertilization potential, so the first ion movements responsible for the fertilization potential are below the resolution of the vibrating probe system. These channels are present in both the animal and vegetal hemispheres, but the magnitude of the activation current is about seven times greater in the animal hemisphere. An outward current of smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of fertilization where the current is first inward. The current direction is dependent on the external Na+ concentration, and in the more physiological solution of 10% NaCl-Yamamoto's Ringer's, its direction reverses to become outward, apparently carried by K+ efflux. Raising the external Ca2+ in this same low-Na+ medium reverses the current so that it becomes inward again and increases the propagation velocity of the wave, suggesting a Ca2+ component to the inward current. Current enters a given region on the egg's surface about 16 sec before any vesicle fusion occurs in that region. Iontophoresis of inositol-1,4,5,-trisphosphate immediately triggers egg activation with a minimum activating charge of 0.6 nC.     

Ion currents and membrane domains in the cleaving Xenopus egg

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.     

Caffeine contracture in the cultured chick myotube

A possible function of Ca store site in cultured chick myotubes was examined by recording contraction of the myotube with special reference to the effect of caffeine. Caffeine at low concentrations (below 1 mM), applied focally on the myotube through a micropipette with a pressure pulse, elicited focal contraction without membrane potential changes. Procaine inhibited the caffeine contracture. Deuterium oxide also inhibited the caffeine contracture at low concentrations, but enhanced the maximal contracture. These observations are similar to those in the mature frog muscle fiber in which the sarcoplasmic reticulum (SR) is a main site of caffeine action. On the basis of these similarities, it was considered that caffeine acts on SR to elicit contracture in the myotube. The ability of SR to accumulate and release Ca ion seemed to be low, because caffeine contracture decreased or disappeared in a Ca-free solution in many myotubes.     

Electrical properties and transmembrane ion currents of single smooth-muscle cells

Current and voltage clamp investigations of freshly isolated smooth muscle cells from guinea-pig ileum and taenia coli were performed using single suction micropipette technique. Specific membrane capacity of smooth muscle cells was calculated and accounted for 1.6 microF/cm2, with specific resistance varying from 50 to 150 k omega X cm2. Transmembrane currents consisted of two inward components, inactivating and noninactivating ones, carried by Ca2+ ions, overlapping with early activated potassium outward current. Time constant of inward current activation was not only voltage-sensitive but also ion-dependent. When Ca2+ ions in Krebs solution were replaced by Ba2+, both the rate of activation and inactivation of inward current were significantly reduced. Estimation of intracellular Ca2+ concentration increase has indicated that inward calcium current transports enough Ca2+ for direct contraction activation.     

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.     

Microelectrode study of K+ accumulation by tight epithelia: I. Baseline values of split frog skin and toad urinary bladder

Toad bladder and split frog skin were impaled with fine-tipped single- and double-barrelled K+-selective microelectrodes. In order to circumvent membrane damage induced by impaling toad bladder, a null point method was developed, involving elevations of mucosal potassium concentration. The results suggest that intracellular potassium activity of short-circuited toad bladder is approximately 82 mM, twice as large as earlier estimates. Far more stable and rigorously defined intracellular measurements were recorded from short-circuited split frog skins. The intracellular positions of the micropipette and microelectrode tips were verified by transient hyperpolarizations of the membrane potential with mucosal amiloride or by transient depolarizations with serosal barium or strophanthidin. Simultaneous impalement of distant cells with separate micropipettes demonstrated that both the baseline membrane potentials and the responses to depolarizing agents were similar, further documenting that frog skin is a functional syncytium. Measurements with double-barrelled microelectrodes and simultaneous single-barrelled microelectrodes and reference micropipettes suggest that the intracellular potassium activity is about 104 mM, lower than previously reported. Taken together with measurements of intracellular potassium concentration, this datum suggests that potassium is uniformly distributed within the epithelial cells.     

Properties of an endogenous steady current in rat muscle

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.     

Localization of ionic pathways in the teleost opercular membrane by extracellular recording with a vibrating probe

We have adapted the vibrating probe extracellular recording technique to use on an epithelium under voltage clamp in an Ussing chamber. The vibrating probe allows very low drift measurements of current density immediately over the epithelial surface. These measurements allowed sites of electrogenic transport in the epithelium to be localized with a spatial resolution of 5 micrometers. The technique was applied to the opercular membrane of the teleost fish, the tilapia, Sarotherodon mossambicus. The mitochondrion-rich "chloride cells" were shown to be the only sites of electrogenic ion transport in this heterogeneous epithelium. Cell sampling experiments demonstrated variable negative short-circuit currents associated with nearly all of approximately 300 chloride cells examined, which appeared to account for all of the tissue short-circuit current. Current-voltage relations for individual cells were also measured. Conductance associated with chloride cells (i.e. cellular and junctional pathways) accounted for all but 0.5 mS/cm2 of the tissue conductance, with the balance apparently accounted for by leak pathways near the edge of the tissue. Current and conductance associated with other cell types was at least 50-fold smaller than for the chloride cell. Chloride-free solutions reduced chloride cell current and conductance by 98 and 95%, respectively.     

Endothelial cell signaling during conducted vasomotor responses

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.     

The cylindrical cell with a time-variant membrane resistance. Measuring passive parameters.

The passive electrical properties of a cable can be measured by injecting a step of current at a point and fitting the resulting potentials at several positions along the cable with analytic solutions of the cable equation. An error analysis is presented for this method (which is based on constant membrane resistance) when the membrane resistance is not constant, but increases linearly with time. The increase of rm produces a "creep" in the membrane potential at long times, as observed in cardiac, skeletal, and smooth muscle. The partial differential equation describing the time-varying cable was solved numberically for a step of current and these "data" were fit by standard constant-resistance methods. Comparing the resulting parameter values with the known true values, we suggest that a correction of the standard methods is not satisfactory for resistance changes of the kind observed; instead, the cable equation must be solved again for the particular form of rm(t). The practical implementation of a method by Adrian and Peachey for measuring the membrane capacitance and an approximate method for estimating the rate-of-change of membrane resistance are discussed in appendices.     

The electrophysiological mapping of compartments within a mammalian cell

The electrical properties of structures within an intact cell were examined by impalement with micropipette electrodes. Mean potential differences (PDs) measured from interphase HeLa cells showed that internal membrane-bounded compartments such as the nucleus, Golgi region, and the mitochondria were more negative than the cytoplasm with respect to an external grounding electrode. The nuclear PDs, unlike Golgi and cytoplasmic PDs, shifted during interphase and reached a peak value shortly before mitosis. The positioning of micropipettes was confirmed by electron microscope examination of marker solutions that were microinjected into specific intracellular regions. The combination of methods described here offers a new approach for the study of physiological events within intact, living cells.     

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.     

The linear electrode array: a useful tool with many applications.

In this review we describe the basic principles of operation of linear electrode arrays for the detection of surface EMG signals, together with their most relevant current applications. A linear array of electrodes is a system which detects surface EMG signals in a number of points located along a line. A spatial filter is usually placed in each point for signal detection, so that the recording of EMG signals with linear arrays corresponds to the sampling in one spatial direction of a spatially filtered version of the potential distribution over the skin. Linear arrays provide indications on motor unit (MU) anatomical properties, such as the locations of the innervation zones and tendons, and the fiber length. Such systems allow the investigation of the properties of the volume conductor and its effect on surface detected signals. Moreover, linear arrays allow to estimate muscle fiber conduction velocity with a very low standard deviation of estimation (of the order of 0.1-0.2 m/s), thus providing reliable indications on muscle fiber membrane properties and their changes in time (for example with fatigue or during treatment). Conduction velocity can be estimated from a signal epoch (global estimate) or at the single MU level. In the latter case, MU action potentials are identified from the interference EMG signals and conduction velocity is estimated for each detected potential. In this way it is possible, in certain conditions, to investigate single MU control and conduction properties with a completely non-invasive approach. Linear arrays provide valuable information on the neuromuscular system properties and appear to be promising tools for applied studies and clinical research.     

Localization of the photocurrent of Limulus ventral photoreceptors using a vibrating probe.

We have used a vibrating probe to determine the profile of electrical current density around ventral photoreceptors of the horseshoe crab following flashes of light that uniformly illuminated the entire surface of the photoreceptor's cell body. The vibrating probe signal indicated that the density of inward current was greatest at the distal region of the cell, the region that is expected to contain the light-sensitive rhabdom. The density of inward current typically declined at the midpoint of the cell body and then reversed to an outward current flow in the proximal region of the cell body, close to the axon. The profile of local sensitivity of the photoreceptor to light closely matched the profile of inward current density, suggesting that the light-activated conductance is localized to the light-sensitive region of the cell.     

The pulse current pattern generated by developing fucoid eggs

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.     

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.     

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to “drive” cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range −200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.     

Development of a steady electric current in neonatal rat lumbrical muscle

We have used a vibrating probe and intracellular recording techniques to study the development of a steady electric current generated by rat lumbrical muscle. In adult animals, previous work has revealed a steady outward current generated at the end plate region. In the present study, we show that at birth muscles generate a steady inward, not outward current. The inward current declines with age, disappearing about 5 days after birth. At about the same time, the steady outward current appears, and reaches adult amplitude by 2-3 weeks after birth. The two currents are generated by completely different mechanisms. The inward current is blocked by alpha-bungarotoxin and apparently results from activation of acetylcholine-gated channels at the end plate. The outward current, on the other hand, is not affected by alpha-bungarotoxin but is blocked by agents which interfere with chloride movements across the membrane, as in the adult.     

Local and remote arteriolar dilations initiated by skeletal muscle contraction

To investigate the relationship between skeletal muscle metabolism and arteriolar dilations in the region local to contracting muscle fibers as well as dilations at remote arteriolar regions upstream, we used a microelectrode on cremaster muscle of anesthetized hamsters to stimulate four to five muscle fibers lying approximately perpendicular to and overlapping a transverse arteriole. Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a remote site approximately 1,000 microm upstream. Two minutes of 2-, 4-, or 8-Hz stimulation (5-10 V, 0.4-ms duration) produced a significant dilation locally (8.2 +/- 2.0-, 22.5 +/- 2.4-, and 30.9 +/- 2.1-microm increase, respectively) and at the remote site (4.2 +/- 0.8, 11.0 +/- 1.1, and 18.9 +/- 2.7 microm, respectively). Muscle contraction at 4 Hz initiated a remote dilation that was unaffected by 15-min micropipette application of either 2 microM tetrodotoxin, 0.07% halothane, or 40 microM 18-beta-glycyrrhetinic acid between the local and upstream site. Therefore, at the arteriolar level, muscle contraction initiates a robust remote dilation that does not appear to be transmitted via perivascular nerves or gap junctions.