Electrical properties and structure of the frog arachnoid membrane

We have used an in vitro preparation of the frog arachnoid membrane to study the role of this membrane in the maintenance of the “blood-cerebrospinal fluid (csf) barrier.” Electron microscopy showed that the membrane was made up of 10–15 layers of flat epithelial cells joined together by numerous cell junctions. The electrical resistance of the preparation was about 2000 ohms cm². The steady-state transmural potential difference (pd) ranged up to 45 mV, csf positive, and this was eliminated by either the addition of ouabain to the csf, or by replacing the NaCl with TEA Cl. The pd across the membrane increased when bicarbonate was added to the external bathing solutions. We conclude that this pd is due to the active transport of sodium from the subural fluid to the csf. In some preparations the transmural pd was reversed, i.e., csf negative, and this was also abolished by the addition of ouabain to the csf, or by replacing chloride with isethionate. We conclude that this pd is related to active chloride transport. These, and other experiments, lead us to the conclusion that the arachnoid membrane is involved in the production of the cerebrospinal fluid and the maintenance of the blood-cerebrospinal fluid barrier.     

Fused cells of frog proximal tubule: I. Basic membrane properties

Summary Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca²⁺ and voltage activated, has a conductance of 221±7 pS, and can be inhibited by 10mm tetraethylammonium and by 1mm quinidine, but not by 4-aminopyridine, nor by 1mm Ba²⁺ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1±3.9 nSvs. a leak conductance of 1.01±0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5±1.4 pF.     

Electrical properties of frog skeletal muscle fibers interpreted with a mesh model of the tubular system.

This paper presents the construction, derivation, and test of a mesh model for the electrical properties of the transverse tubular system (T-system) in skeletal muscle. We model the irregular system of tubules as a random network of miniature transmission lines, using differential equations to describe the potential between the nodes and difference equations to describe the potential at the nodes. The solution to the equations can be accurately represented in several approximate forms with simple physical and graphical interpretations. All the parameters of the solution are specified by impedance and morphometric measurements. The effect of wide circumferential spacing between T-system openings is analyzed and the resulting restricted mesh model is shown to be approximated by a mesh with an access resistance. The continuous limit of the mesh model is shown to have the same form as the disk model of the T-system, but with a different expression for the tortuosity factor. The physical meaning of the tortuosity factor is examined, and a short derivation of the disk model is presented that gives results identical to the continuous limit of the mesh model. Both the mesh and restricted mesh models are compared with experimental data on the impedance of muscle fibers of the frog sartorius. The derived value for the resistivity of the lumen of the tubules is not too different from that of the bathing solution, the difference probably arising from the sensitivity of this value to errors in the morphometric measurements.     

Electrical properties and gustatory responses of various taste disk cells of frog fungiform papillae

We compared the electrical properties and gustatory response profiles of types Ia cell (mucus cell), Ib cell (wing cell), and II/III cell (receptor cell) in the taste disks of the frog fungiform papillae. The large depolarizing responses of all types of cell induced by 1 M NaCl were accompanied by a large decrease in the membrane resistance and had the same reversal potential of approximately +5 mV. The large depolarizing responses of all cell types for 1 mM acetic acid were accompanied by a small decrease in the membrane resistance. The small depolarizing responses of all cell types for 10 mM quinine-HCl (Q-HCl) were accompanied by an increase in the membrane resistance, but those for 1 M sucrose were accompanied by a decrease in the membrane resistance. The reversal potential of sucrose responses in all cell types were approximately +12 mV. Taken together, depolarizing responses of Ia, Ib, and II/III cells for each taste stimulus are likely to be generated by the same mechanisms. Gustatory depolarizing response profiles indicated that 1) each of Ia, Ib, and II/III cells responded 100% to 1 M NaCl and 1 mM acetic acid with depolarizing responses, 2) approximately 50% of each cell type responded to 10 mM Q-HCl with depolarizations, and 3) each approximately 40% of Ia and Ib cells and approximately 90% of II/III cells responded to 1 M sucrose with depolarizations. These results suggest that the receptor molecules for NaCl, acid, and Q-HCl stimuli are equivalently distributed on all cell types, but the receptor molecules for sugar stimuli are richer on II/III cells than on Ia and Ib cells. Type III cells having afferent synapses may play a main role in gustatory transduction and transmission.     

Electrical properties of the myotendon region of frog twitch muscle fibers measured in the frequency domain

The electrical properties of the end of a muscle fiber were determined using three microelectrodes, one passing sinusoidal current, the other two recording the resulting voltages. An electrical model was constructed from the morphology of the fiber, including the resistance of the extracellular space between cells; the parameters of this model were determined by fitting the model to the observed voltage responses. Our results, analyzed directly or by curve fits, show that the end of muscle fibers contains a large capacitance resulting from the extensive membrane folds at the myotendon junction. Analysis and simulations show that the extra capacitance at the myotendon junction has substantial effects on measurements of linear properties, in particular on estimates of the capacitance of the membranes. There is little qualitative effect on classical measurements of nonlinear charge movement (provided they were made with one set of electrode locations) if the linear components have been subtracted. Quantitative estimates of nonlinear charge movement and ionic currents are significantly affected, however, because these estimates are customarily normalized with respect to the linear capacitance.     

Electrical properties of plasma membrane modulate subcellular distribution of K-Ras

K-Ras is a small G-protein, localized mainly at the inner leaflet of the plasma membrane. The membrane targeting signal of this protein consists of a polybasic C-terminal sequence of six contiguous lysines and a farnesylated cysteine. Results from biophysical studies in model systems suggest that hydrophobic and electrostatic interactions are responsible for the membrane binding properties of K-Ras. To test this hypothesis in a cellular system, we first evaluated in vitro the effect of electrolytes on K-Ras membrane binding properties. Results demonstrated the electrical and reversible nature of K-Ras binding to anionic lipids in membranes. We next investigated membrane binding and subcellular distribution of K-Ras after disruption of the electrical properties of the outer and inner leaflets of plasma membrane and ionic gradients through it. Removal of sialic acid from the outer plasma membrane caused a redistribution of K-Ras to recycling endosomes. Inhibition of polyphosphoinositide synthesis at the plasma membrane, by depletion of cellular ATP, resulted in a similar subcellular redistribution of K-Ras. Treatment of cells with ionophores that modify transmembrane potential caused a redistribution of K-Ras to cytoplasm and endomembranes. Ca2+ ionophores, compared to K+ ionophores, caused a much broader redistribution of K-Ras to endomembranes. Taken together, these results reveal the dynamic nature of interactions between K-Ras and cellular membranes, and indicate that subcellular distribution of K-Ras is driven by electrostatic interaction of the polybasic region of the protein with negatively charged membranes.     

Electrical characteristics of frog atrial trabeculae in the double sucrose gap.

The electrical behavior of small single frog atrial trabeculae in the double sucrose gap has been investigated. The currents injected during voltage clamp experiments did not behave as predicted from the assumption of spatial uniformity of the voltage across a Hodgkin-Huxley membrane. Much of the difference is due to the geometrical complexities of this tissue. Nonetheless, two transient inward currents have been identified, the faster of which is blocked by tetrodotoxin (TTX). The magnitude of the slower transient varies markedly between preparations but always increases in a given preparation with increase of external calcium. The fast transient current traces, at small to intermediate depolarizations, are often marred by the presence of notches and secondary peaks due most probably to the loss of space clamp conditions. In many preparations these could be removed by reducing the current magnitude through application of a partially-blocking dose of TTX. Conversely, in the preparations whose fast transient was fully blocked by TTX, notches and secondary peaks in the slow transient could by induced through increasing calcium concentration and thereby the slow current magnitude. Previously used techniques for the measurement of the reversal potential of the fast inward transient have been shown to be invalid. In so far as they can be measured, the reversal potentials of the fast and slow inward transient are in the same neighborhood, i.e. around 120 mV from rest. The true values may be quite a bit apart. The total charge flow in the capacitive transient was measured for different sized nodes and preparations. From these data and estimates of plasma membrane area per unit trabecular volume, specific membrane capacitances of around 3 muF/cm2 were calculated for small bundles. The apparent ion current densities on this basis are approximately 1/10 of those measured in axons. The capacitive current occurring in small bundles decayed as the sum of at least three exponential functions of time. On the basis of these data and the anomalously large stable node widths, we suggest a coaxial core model of the preparation with the inner elements in series with an additional large extracellular resistance.     

Structural properties of frog muscle myosin.

Frog myosin is a labile molecule, undergoing irreversible aggregation and rapid loss of ATPase; however, a procedure is described which provides highly purified myosin, with stable solubility and enzymatic properties, from skeletal muscle of Rana catesbeiana. Frog myosin contains heavy chains and light chains 1, 2, and 3. Light chain 3 is present in excess over light chain 1, and light chain 2 may occur as either, or both, of 2 closely migrating bands. On two-dimensional electrophoresis, light chain 1 generates an isoelectric component with pK 5.60; light chain 2 generates a complex pattern with 3 or 4 major components; and light chain 3 generates 2 major components with pK 5.00 and 4.92. The same subunit composition is obtained for frogs acclimated at 25 and 5 degrees C; however, proteolytic artifacts may occur in myosin preparations purified in the absence of protease inhibitors, especially in warm-acclimated frogs.     

Electrical and mechanical activity of isoproterenol-damaged frog heart

The electrical and mechanical activities of myocardial strips from Rana pipiens were studied in the steady state at various stimulation frequencies and after a period of rest. The temperature of the bath was varied between + 12 and 35 degrees C. Normal myocardium was compared with that damaged by isoproterenol (ISO). The percentage change of action potential durations (APD50) and isometric force (P) was similar in ISO-damaged and control hearts at various bath temperatures with steady state stimulation rates between 20 and 100/min. At low stimulation rates (3-6/min) the prolongation of the action potential (AP) was more pronounced and the P-decrease was less in ISO-damaged myocardium compared to controls. These differences became more apparent at low bath temperatures. After 10 min of rest, APD50-90 was significantly prolonged in ISO-damaged heart and P was increased by a factor of 2 compared to controls (stimulation rate 20/min). Steady state values, on the other hand, were nearly the same in both groups. These findings are interpreted as indicating a temperature or ISO-dependent increase of electrogenic trans-sarcolemmal Ca2+-uptake during low frequency, or post-rest stimulation, either directly by an increase of the slow inward current (Isi), or indirectly by decreased K+-permeabilities.     

Nucleus isthmi of the frog: structure and tecto-isthmic projection.

The morphology of neurons in the isthmic nucleus was studied with the Golgi technique. Most of the neurons have thick dendrites covered with lamelliform dendritic processes. The tecto-isthmic projection was investigated with the Fink--Heimer technique after partial tectal lesions. The anteromedial part of the tectum projects on the dorsal and anterior part, the caudomedial tectal region on the dorsal and posterior part of the nucleus. The posterolateral tectal area projects on the anterolateral part of the nucleus, and the axons originating in the anterolateral tectal quadrant terminate in its ventral and caudal part.     

Electrical properties of the cellular transepithelial pathway in Necturus gallbladder. II. Ionic permeability of the apical cell membrane.

Microelectrode techniques were employed to study the ionic permeability of the apical cell membrane of Necturus gallbladder epithelium. Results obtained from continuous records in single cells, and from several cellular impalements shortly after a change in solution, were similar and indicate that both the apical membrane equivalent electromotive force (Va) and electrical resistance (Ra) strongly depend on external [K]. Cl substitutions produced smaller effects, while the effects of Na substitutions with N-methyl-D-glucamine on both Va and Ra were minimal. These results indicate that the permeability sequence of the apical membrane is PKgreater thanPClgreater than PNa. From the calculated absolute value of PNa it is possible to estimate the diffusional Na flux from the mucosal solution into the cells (from the cell potential and an assumed intracellular Na concentration). The calculated flux is roughly three orders of magnitude smaller than the measured net transepithelial flux in this tissue and in gallbladders of other species. Thus, only a minimal portion of Na entry can be attributed to independent diffusion. From estimations of the electrochemical potential gradient across the apical membrane, Cl transport at that site must be active. At the serosal cell membrane, Na transport takes place against both chemical and electrical potentials, while a significant portion of the Cl flux can be passive, if this membrane has a significant Cl conductance. The changes in shunt electromotive force and in transepithelial potential after mucosal substitutions were very similar, indicating that transepithelial bi-ionic potentials yield appropriate results on the properties of shunt pathway.