Multi-ion conduction bands in a simple model of calcium ion channels.

We report self-consistent Brownian dynamics simulations of a simple electrostatic model of the selectivity filters (SF) of calcium ion channels. They reveal regular structure in the conductance and selectivity as functions of the fixed negative charge Qf at the SF. With increasing Qf, there are distinct regions of high conductance (conduction bands) M0, M1, M2 separated by regions of almost zero-conductance (stop-bands). Two of these conduction bands, M1 and M2, are related to the saturated calcium occupancies of P = 1 and P = 2, respectively and demonstrate self-sustained conductivity. Despite the model's limitations, its M1 and M2 bands show high calcium selectivity and prominent anomalous mole fraction effects and can be identified with the L-type and RyR calcium channels. The non-selective band M0 can be identified with a non-selective cation channel, or with OmpF porin.

Structure and function in gene patenting.

The United States patent system treats DNA sequences as large chemical compounds in determining their patentability. This approach has been helpful to those who seek to patent previously unidentified DNA sequences, but it may prove less advantageous from the perspective of those who elucidate biological functions and disease relevance of previously identified genes. A current controversy over patent rights for DNA sequences encoding leptin receptors provides a useful case study for illustrating some of the issues that are likely to arise in applying doctrine derived from chemical patent cases in the context of gene discovery.

Selective disruption of the sarcotubular system in frog sartorius muscle. A quantitative study with exogenous peroxidase as a marker.

Skeletal muscles which have been soaked for 1 hr in a glycerol-Ringer solution and then returned to normal Ringer solution have a disrupted sarcotubular system. The effect is associated with the return to Ringer's since muscles have normal fine structure while still in glycerol-Ringer's. Karnovsky's peroxidase method was found to be a very reliable marker of extracellular space, filling 98.5% of the tubules in normal muscle. It was interesting to note that only 84.1% of the sarcomeres in normal muscle have transverse tubules. The sarcotubular system was essentially absent from glycerol-treated muscle fibers, only 2 % of the tubular system remaining connected to the extracellular space; the intact remnants were stumps extending only a few micra into the fiber. Thus, glycerol-treated muscle fibers provide a preparation of skeletal muscle with little sarcotubular system. Since the sarcoplasmic reticulum is not destroyed and the sarcolemma and myofilaments are intact in this preparation, of the properties of the sarcolemma may thus be separated from those of the tubular system.

Genes, patents, and product development.

In the past year, the National Institutes of Health (NIH) has filed patent applications on more than 2750 partial complementary DNA sequences of unknown function. The rationale for the filings--that patent protection may be necessary to ensure that private firms are willing to invest in developing related products--rests on two premises: first, that NIH may obtain patent rights that will offer effective product monopolies to licensee firms, and second, that unless NIH obtains these rights now, firms will be unable to obtain a comparable degree of exclusivity by other means, such as by obtaining patents on their own subsequent innovations. Neither premise is clearly wrong, although both are subject to doubt in view of statements from industry representatives that the NIH patenting strategy will deter rather than promote product development.

Transverse tubular system in glycerol-treated skeletal muscle.

Horseradish peroxidase used as an extracellular marker fills 98.5 percent of the central triadic elements (tubules) in normal muscle and 97.2 percent in muscle soaked in Ringer solution to which 400-millimolar glycerol is added. A glycerol-soaked muscle rapidly returned to normal Ringer solution has a disrupted transverse tubular system and has only 3.2 percent of triads filled with peroxidase.

Can patents deter innovation? The anticommons in biomedical research.

The "tragedy of the commons" metaphor helps explain why people overuse shared resources. However, the recent proliferation of intellectual property rights in biomedical research suggests a different tragedy, an "anticommons" in which people underuse scarce resources because too many owners can block each other. Privatization of biomedical research must be more carefully deployed to sustain both upstream research and downstream product development. Otherwise, more intellectual property rights may lead paradoxically to fewer useful products for improving human health.

Action potentials without contraction in frog skeletal muscle fibers with disrupted transverse tubules.

Action potentials, with no accompanying contraction, were recorded from muscle fibers in which the transverse tubular system had been disrupted. The results show that action potentials require an intact transverse tubular system to cause contraction. Furthermore, both the after-depolarization following a single action potential and the slower, late afterpotential following a train of action potentials were absent in this preparation. Therefore, both phenomena must normally involve the transverse tubular system.

Frog skeletal muscle fibers: changes in electrical properties after disruption of transverse tubular system.

In muscle fibers which have been exposed for 1 hour to a Ringer solution containing 400 millimolar glycerol and then returned to plain Ringer solution, the transverse tubular system is disrupted. At the same time the membrane capacitance is markedly reduced and hyperpolarizing current pulses no longer produce a slow, progressive increase in potential (creep). The large capacitance of muscle and the phenomenon of "creep" must both depend on an intact transverse tubular system.

Self-consistent analytic solution for the current and the access resistance in open ion channels.

A self-consistent analytic approach is introduced for the estimation of the access resistance and the current through an open ion channel for an arbitrary number of species. For an ion current flowing radially inward from infinity to the channel mouth, the Poisson-Boltzmann-Nernst-Planck equations are solved analytically in the bulk with spherical symmetry in three dimensions, by linearization. Within the channel, the Poisson-Nernst-Planck equation is solved analytically in a one-dimensional approximation. An iterative procedure is used to match the two solutions together at the channel mouth in a self-consistent way. It is shown that the current-voltage characteristics obtained are in good quantitative agreement with experimental measurements.

Integrated electrodes on a silicon based ion channel measurement platform.

We demonstrate the microfabrication of a low-noise silicon based device with integrated silver/silver chloride electrodes used for the measurement of single ion channel proteins. An aperture of 150 microm diameter was etched in a silicon substrate using a deep silicon reactive ion etcher and passivated with 30 nm of polytetrafluoroethylene via chemical vapor deposition. The average recorded noise in measurements of lipid bilayers was reduced by a factor of four through patterning of a 75 microm thick SU-8 layer around the aperture. Integrated electrodes were fabricated on both sides of the device and used for repeatable, stable, giga-seal bilayer formations as well as characteristic measurements of the transmembrane protein OmpF porin.

The equivalent circuit of single crab muscle fibers as determined by impedance measurements with intracellular electrodes.

The input impedance of muscle fibers of the crab was determined with microelectrodes over the frequency range 1 cps to 10 kc/sec. Care was taken to analyze, reduce, and correct for capacitive artifact. One dimensional cable theory was used to determine the properties of the equivalent circuit of the membrane admittance, and the errors introduced by the neglect of the three dimensional spread of current are discussed. In seven fibers the equivalent circuit of an element of the membrane admittance must contain a DC path and two capacitances, each in series with a resistance. In two fibers, the element of membrane admittance could be described by one capacitance in parallel with a resistance. In several fibers there was evidence for a third very large capacitance. The values of the elements of the equivalent circuit depend on which of several equivalent circuits is chosen. The circuit (with a minimum number of elements) that was considered most reasonably consistent with the anatomy of the fiber has two branches in parallel: one branch having a resistance R(e) in series with a capacitance C(e); the other branch having a resistance R(b) in series with a parallel combination of a resistance R(m) and a capacitance C(m). The average circuit values (seven fibers) for this model, treating the fiber as a cylinder of sarcolemma without infoldings or tubular invaginations, are R(e) = 21 ohm cm(2); C(e) = 47 microf/cm(2); R(b) = 10.2 ohm cm(2); R(m) = 173 ohm cm(2); C(m) = 9.0 microf/cm(2). The relation of this equivalent circuit and another with a nonminimum number of circuit elements to the fine structure of crab muscle is discussed. In the above equivalent circuit R(m) and C(m) are attributed to the sarcolemma; R(e) and C(e), to the sarcotubular system; and R(b), to the amorphous material found around crab fibers. Estimates of actual surface area of the sarcolemma and sarcotubular system permit the average circuit values to be expressed in terms of unit membrane area. The values so expressed are consistent with the dielectric properties of predominantly lipid membranes.

The T-SR junction in contracting single skeletal muscle fibers.

The junction between the T system and sarcoplasmic reticulum (SR) of frog skeletal muscle was examined in resting and contracting muscles. Pillars, defined as pairs of electron-opaque lines bounding an electron-lucent interior, were seen spanning the gap between T membrane and SR. Feet, defined previously in images of heavily stained preparations, appear with electron-opaque interiors and as such are distinct from the pillars studied here. Amorphous material was often present in the gap between T membrane and SR. Sometimes the amorphous material appeared as a thin line parallel to the membranes; sometimes it seemed loosely organized at the sites where feet have been reported. Resting single fibers contained 39 +/- 14.3 (mean +/- SD; n = 9 fibers) pillars/micrometer2 of tubule membrane. Single fibers, activated by a potassium-rich solution at 4 degrees C, contained 66 +/- 12.9 pillars/micrometer2 (n = 8) but fibers contracting in response to 2 mM caffeine contained 33 +/- 8.6/micrometer2 (n = 5). Pillar formation occurs when fibers are activated electrically, but not when calcium is released directly from the SR; and so we postulate that pillar formation is a step in excitation-contraction coupling.

Calcium influx in contracting and paralyzed frog twitch muscle fibers.

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.

Capacitance of the surface and transverse tubular membrane of frog sartorius muscle fibers.

The passive electrical properties of glycerol-treated muscle fibers, which have virtually no transverse tubules, were determined. Current was passed through one intracellular microelectrode and the time course and spatial distribution of the resulting potential displacement measured with another. The results were analyzed by using conventional cable equations. The membrane resistance of fibers without tubules was 3759 +/- 331 ohm-cm(2) and the internal resistivity 192 ohm-cm. Both these figures are essentially the same as those found in normal muscle fibers. The capacitance of the fibers without tubules is strikingly smaller than normal, being 2.24 +/- 0.14 microF/cm(2). Measurements were also made of the passive electrical properties of fibers in a Ringer solution containing 400 mM glycerol (which is used in the preparation of glycerol-treated fibers). The membrane resistance and capacitance are essentially normal, but the internal resistivity is somewhat reduced. These results show that glycerol in this concentration does not directly affect the membrane capacitance. Thus, the figure for the capacitance of glycerol-treated fibers, which agrees well with previous estimates made by different techniques, represents the capacitance of the outer membrane of the fiber. Estimates of the capacitance per unit area of the tubular membrane are made and the significance of the difference between the figures for the capacitance of the surface and tubular membrane is discussed.

Ionic conductances of the surface and transverse tubular membranes of frog sartorius fibers.

The resting ionic conductances of frog sartorius muscle fibers have been determined in a variety of conditions in order to measure the potassium conductance of the tubular and surface membranes (gK(t) and gK(s)) and the chloride conductance of the tubular and surface membranes (gCl(t) and gCl(s)). In both normal fibers and fibers without tubules, measurements of input resistance and diameter were made at normal pH and at low pH when the chloride conductance was very small. These measurements permitted the separation of the ionic conductances: gCl(s) = 219 micromhos/cm(2); gCl(t) = 0 micromhos/cm(2); gK(s) = 28 micromhos/cm(2); gK(t) = 55 micromhos/cm(2). Possible sources of systematic error are discussed and a statistical analysis of the effects of random error is presented. The implications of the nonuniformity of membrane properties are discussed along with possible anatomical explanations.

Action potentials, afterpotentials, and excitation-contraction coupling in frog sartorius fibers without transverse tubules.

In frog sartorius muscle fibers in which the transverse tubular system has been disrupted by treatment with glycerol, action potentials which are unaccompanied by twitches can be recorded. These action potentials appear to be the same as those recorded in normal fibers except that the early afterpotential usually consists of a small hyperpolarization of short duration. After a train of action potentials no late afterpotential is seen even when the membrane potential is changed from the resting level. In fibers without transverse tubules hyperpolarizing currents do not produce a creep in potential. The interruption of excitation-contraction coupling, the changes in the afterpotentials, and the disappearance of creep are all attributed to the lack of a transverse tubular system.

The spatial variation of membrane potential near a small source of current in a spherical cell.

A theoretical analysis is presented of the change in membrane potential produced by current supplied by a microelectrode inserted just under the membrane of a spherical cell. The results of the analysis are presented in tabular and graphic form for three wave forms of current: steady, step function, and sinusoidal. As expected from physical reasoning, we find that the membrane potential is nonuniform, that there is a steep rise in membrane potential near the current microelectrode, and that this rise is of particular importance when the membrane resistance is low, or the membrane potential is changing rapidly. The effect of this steep rise in potential on the interpretation of voltage measurements from spherical cells is discussed and practical suggestions for minimizing these effects are made: in particular, it is pointed out that if the current and voltage electrodes are separated by 60 degrees , the change in membrane potential produced by application of current is close to that which would occur if there were no spatial variation of potential. We thus suggest that investigations of the electrical properties of spherical cells using two microelectrodes can best be made when the electrodes are separated by 60 degrees .

Longitudinal impedance of single frog muscle fibers.

The longitudinal impedance of single skeletal muscle fibers has been measured from1 to 10,000 Hz in an oil gap apparatus which forces current to flow longitudinally down the fiber. The impedance observed is purely resistive in some fibers from the semitendinosus muscle and in two fibers from the sartorius muscle. In other fibers from the semitendinosus muscle a small phase shift is observed. The mean value of the maximum phase shift observed from all fibers is 1.07 degrees. The artifacts associated with the apparatus and method are examined theoretically and it is shown that one of the likely artifacts could account for the small phase observed. It is concluded that the longitudinal impedance of skeletal muscle fibers is essentially resistive and that little, if any, longitudinal current crosses the membranes of the sarcoplasmic reticulum.

Longitudinal impedance of skinned frog muscle fibers.

Longitudinal impedance of skinned muscle fibers was measured with extracellular electrodes and an oil gap method in which a central longitudinal section of fiber is insulated by oil while the ends of the fiber are bathed in conducting pools of relaxing solution. Intact single fibers were isolated from frog semitendinosus muscle and the sarcolemma removed either by mechanical or chemical methods. Stray capacitance across the oil gap was measured after each experiment and its admittance subtracted from the admittance of the fiber and oil gap. Effects of impedance at the ends of the fiber were eliminated by measuring the impedance with two lengths of fiber in the oil gap and subtracting the impedance at the shorter length from that at the longer length. Longitudinal impedance so determined for mechanically and chemically skinned fibers exhibited zero phase shift from 1 to 10,000 Hz, i.e., the longitudinal impedance of skinned fibers is purely resistive. If we assume that our skinned fibers are a model of the sarcoplasm of muscle, we conclude that the equivalent circuit of the sarcoplasm is a resistor.