Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP

A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.     

Fast inactivation causes rectification of the IKr channel

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current- voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.     

Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.     

Gating of delayed rectification in acutely isolated canine cardiac Purkinje myocytes. Evidence for a single voltage-gated conductance.

Studies of time-dependent, plateau outward current (delayed rectification) in the heart are complicated by the accumulation and depletion of K+ ions in intercellular clefts. To minimize this problem, we studied delayed rectification in acutely isolated (enzymic solution, gentle agitation) canine cardiac Purkinje myocytes using the single microelectrode voltage-clamp technique. We found a sigmoidal voltage-dependence for activation of outward plateau current, with maximal activation occurring at potentials near -10 mV. The activation and deactivation of plateau outward current was adequately described as the sum of a fast and slow exponential component. A comparison of the time course of activation of plateau outward current and the "envelope" of tail currents suggests that a single voltage-gated conductance with one open and two closed states can account for delayed rectification in Purkinje myocytes. These results differ from those previously obtained with intact sheep Purkinje fibers in which two time-dependent conductances were postulated to account for delayed rectification (Noble, D., and R. W. Tsien, 1969, J. Physiol. (Lond.), 200:205-231).     

Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents

An envelope of tails test was used to show that the delayed rectifier K+ current (IK) of guinea pig ventricular myocytes results from the activation of two outward K+ currents. One current was specifically blocked by the benzenesulfonamide antiarrhythmic agent, E-4031 (IC50 = 397 nM). The drug-sensitive current, "IKr" exhibits prominent rectification and activates very rapidly relative to the slowly activating drug-insensitive current, "IKs." IKs was characterized by a delayed onset of activation that occurs over a voltage range typical of the classically described cardiac IK. Fully activated IKs, measured as tail current after 7.5-s test pulses, was 11.4 times larger than the fully activated IKr. IKr was also blocked by d-sotalol (100 microM), a less potent benzenesulfonamide Class III antiarrhythmic agent. The activation curve of IKr had a steep slope (+7.5 mV) and a negative half-point (-21.5 mV) relative to the activation curve of IKs (slope = +12.7 mV, half-point = +15.7 mV). The reversal potential (Erev) of IKr (-93 mV) was similar to EK (-94 mV for [K+]o = 4 mM), whereas Erev of IKs was -77 mV. The time constants for activation and deactivation of IKr made up a bell-shaped function of membrane potential, peaking between -30 and -40 mV (170 ms). The slope conductance of the linear portion of the fully activated IKr-V relation was 22.5 S/F. Inward rectification of this relation occurred at potentials greater than -50 mV, resulting in a voltage-dependent decrease in peak IKr at test potentials greater than 0 mV. Peak IKr at 0 mV averaged 0.8 pA/pF (n = 21). Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (225 ms) at membrane potentials (-20 to +20 mV) typical of the plateau phase of cardiac action potentials.     

Inverted Current–Voltage Hysteresis in Mixed Perovskite Solar Cells: Polarization,Energy Barriers,and Defect Recombination

Organic‐inorganic metal halide perovskite solar cells show hysteresis in their current–voltage curve measured at a certain voltage sweep rate. Coinciding with a slow transient current response, the hysteresis is attributed to a slow voltage‐driven (ionic) charge redistribution in the perovskite solar cell. Thus, the electric field profile and in turn the electron/hole collection efficiency become dependent on the biasing history. Commonly, a positive prebias is beneficial for a high power‐conversion efficiency. Fill factor and open‐circuit voltage increase because the prebias removes the driving force for charge to pile‐up at the electrodes, which screen the electric field. Here, it is shown that the piled‐up charge can also be beneficial. It increases the probability for electron extraction in case of extraction barriers due to an enhanced electric field allowing for tunneling or dipole formation at the perovskite/electrode interface. In that case, an inverted hysteresis is observed, resulting in higher performance metrics for a voltage sweep starting at low prebias. This inverted hysteresis is particularly pronounced in mixed‐cation mixed‐halide systems which comprise a new generation of perovskite solar cells that makes it possible to reach power‐conversion efficiencies beyond 20%.     

Steady-state availability of sodium channels. Interactions between activation and slow inactivation.

Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.     

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.     

Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating [published erratum appears in J Gen Physiol 1989 Aug;94(2):401]

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.     

Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes.

Ferret atrial myocytes can display an E-4031-sensitive current (IKr) that is similar to that previously described for guinea pig cardiac myocytes. We examined the ferret atrial IKr as the E-4031-sensitive component of current using the amphotericin B perforated patch-clamp technique. Steady-state IKr during depolarizing pulses showed characteristic inward rectification. Activation time constants during a single pulse were voltage dependent, consistent with previous studies. However, for potentials positive to +30 mV, IKr time course became complex and included a brief transient component. We examined the envelope of tails of the drug-sensitive current for activation in the range -10 to +50 mV and found that the tail currents for IKr do not activate with the same time course as the current during the depolarizing pulse. The activation time course determined from tail currents was relatively voltage insensitive over the range +30 to +50 mV (n = 5), but was voltage sensitive for potentials between -10 and +30 mV and appeared to show some sigmoidicity in this range. These data indicate that activation of IKr occurs in at least two steps, one voltage sensitive and one voltage insensitive, the latter of which becomes rate limiting at positive potentials. We also examined the rapid time-dependent inactivation process that mediates rectification at positive potentials. The time constants for this process were only weakly voltage dependent over the range of potentials from -50 to +60 mV. From these data we constructed a simple linear four-state model that reproduces the general features of ferret IKr, including the initial transient at positive potentials and the apparent discrepancy between the currents during the initial depolarizing pulse and the tail current.     

Distribution and kinetics of membrane dielectric polarization. 1. Long- term inactivation of gating currents

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.     

A slow outward current and a hypoosmolality induced anion conductance in embryonic chicken osteoclasts

In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) > P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

Cholesterol Accumulation Increases Insulin Granule Size and Impairs Membrane Trafficking

The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic β cells, cholesterol accumulation causes defects in insulin secretion and may participate in the pathogenesis of type 2 diabetes. Using a novel cholesterol analog, we show for the first time that insulin granules are the major sites of intracellular cholesterol accumulation in live β cells. This is distinct from other, non‐secretory cell types, in which cholesterol is concentrated in the recycling endosomes and the trans‐Golgi network. Excess cholesterol was delivered specifically to insulin granules, which caused granule enlargement and retention of syntaxin 6 and VAMP4 in granule membranes, with concurrent depletion of these proteins from the trans‐Golgi network. Clathrin also accumulated in the granules of cholesterol‐overloaded cells, consistent with a possible defect in the last stage of granule maturation, during which clathrin‐coated vesicles bud from the immature granules. Excess cholesterol also reduced the docking and fusion of insulin granules at the plasma membrane. Together, the data support a model in which cholesterol accumulation in insulin secretory granules impairs the ability of these vesicles to respond to stimuli, and thus reduces insulin secretion.     

Extracellular calcium transients at single excitations in rabbit atrium measured with tetramethylmurexide

Extracellular calcium transients were resolved within the time course of single contraction cycles in rabbit left atrium using tetramethylmurexide (2 mM) as the calcium-sensitive dye (150-250 microM total calcium, 80-150 microM free calcium). Net extracellular calcium depletion began within 2-4 ms upon excitation; over the following 5-20 ms, depletion continued steeply and amounted to 0.2 mumol/kg wet weight X 10 ms (135 microM free extracellular calcium). In regularly excited muscles (0.5-2 Hz), net depletion slowed rapidly and stopped early during the rise of contractile motion monitored by transmitted light. Maximum depletions amounted to 0.2-0.5% of total extracellular calcium (0.2-0.5 mumol/kg wet weight with 135 microM free calcium). Replenishment of extracellular calcium began at the latest midway to the peak of the motion signal. Calcium replenishment could be complete for the most part by an early phase of relaxation or could take place continuously through relaxation. The maximal net depletion per beat decreased manyfold with a decrease of frequency from 1 to 0.05 Hz. During paired pulse stimulation (200-300-ms twin pulse separation at basal rates of 0.3-1 Hz), extracellular calcium accumulation was enhanced at the initial potentiated contraction; extracellular calcium depletion was prolonged at the low-level premature contraction. With quadruple stimulation (three premature excitations), the apparent rate of net extracellular calcium accumulation at potentiated contractions approached or exceeded the apparent rate of early net calcium depletion. Under the special circumstance of a strongly potentiated post-stimulatory contraction after greater than 5 s rest, repolarization beyond -40 mV occurred within 10 ms, net extracellular calcium accumulation began with the onset of muscle motion, and net extracellular calcium accumulation (1-3 microM/kg wet weight) coincided with a more positive late action potential in comparison with subsequent action potentials. Consistent changes of the apparent rate of early net calcium depletion were not found with any of the simulation patterns examined. In ryanodine-pretreated atria, the duration of depletion was clearly limited by action potential duration at post-rest stimulations; in the presence of 4-aminopyridine (2 mM), depletion continued essentially undiminished for up to 200 ms. The resulting net depletion magnitudes were greater than 10 times larger than the transient depletions found during steady stimulation.     

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.     

Ion Current Rectification,Limiting and Overlimiting Conductances in Nanopores

Previous reports on Poisson-Nernst-Planck (PNP) simulations of solid-state nanopores have focused on steady state behaviour under simplified boundary conditions. These are Neumann boundary conditions for the voltage at the pore walls, and in some cases also Donnan equilibrium boundary conditions for concentrations and voltages at both entrances of the nanopore. In this paper, we report time-dependent and steady state PNP simulations under less restrictive boundary conditions, including Neumann boundary conditions applied throughout the membrane relatively far away from the nanopore. We simulated ion currents through cylindrical and conical nanopores with several surface charge configurations, studying the spatial and temporal dependence of the currents contributed by each ion species. This revealed that, due to slow co-diffusion of oppositely charged ions, steady state is generally not reached in simulations or in practice. Furthermore, it is shown that ion concentration polarization is responsible for the observed limiting conductances and ion current rectification in nanopores with asymmetric surface charges or shapes. Hence, after more than a decade of collective research attempting to understand the nature of ion current rectification in solid-state nanopores, a relatively intuitive model is retrieved. Moreover, we measured and simulated current-voltage characteristics of rectifying silicon nitride nanopores presenting overlimiting conductances. The similarity between measurement and simulation shows that overlimiting conductances can result from the increased conductance of the electric double-layer at the membrane surface at the depletion side due to voltage-induced polarization charges. The MATLAB source code of the simulation software is available via the website http://micr.vub.ac.be.     

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.