The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates ("flip")

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 μM), openings occurred in long (>300-ms) groups with high open probability (P(open); 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5-10 μs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ～130,000 s(-1), much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum P(open) is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.     

A note on correlations in single ion channel records

General expressions are derived for the correlation coefficients between the length of an opening and that of the nth subsequent opening for a single ion channel. Analogous results are given for the correlation between shut times, and between an open time and subsequent shut times. An alternative derivation of the results of Fredkin et al. (in Proc. Berkeley Conf. in honor of Neyman & Kiefer, vol. 1, pp. 269-289 (1985] is given, and their results are extended to the case where openings occur in bursts. Expressions are given for the correlation between the first and nth opening in a burst, between the lengths of bursts, and between the number of openings per burst. Each of these sorts of correlation can give information about the connections that exist between the various states of the system; interpretations of the correlations are discussed. Expressions are derived for the distributions of the nth open time, shut time, burst length, etc. following the application of a perturbation (e.g. a voltage jump or a concentration jump). It is shown that these distributions will all be the same (namely the equilibrium distribution) only in the case where the openings, burst lengths, etc. are not correlated. Certain reaction schemes predict a component in the distribution of the number of openings per burst that has a unit mean (i.e. a component of isolated single openings). For some schemes this component is predicted to have zero amplitude, in principle, whereas in others it may be quite prominent. The presence or absence of this component can give information about the way in which the various states of the system are connected. The interpretation in terms of mechanism is discussed.     

G-protein-gated TRP-like cationic channel activated by muscarinic receptors: effect of potential on single-channel gating

There is little information about the mechanisms by which G-protein-coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPgammaS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the K(ACh) channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245-253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BK(Ca) channel). Open probability (P(O)) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca(2+) and Galphao-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when P(O) was measured over 5-s intervals. This was related to the variable frequency of openings (as in the K(ACh) channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as P(O) increased 525-fold in three stages.     

Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed.

If a single ion channel record is observed in which two ion channels are never simultaneously open, then it is often of interest to know whether the observations indeed arose from the activity of only one ion channel. This question can be answered if it is possible to calculate the distribution of the duration of runs of single openings in a membrane patch that contains two active channels. If the observed run of single openings is much longer than that expected for a patch with two channels it is likely that only one channel was active. An approximate method is presented for calculating the distribution of the duration of runs of single openings in a patch with two active channels; this method has the advantage that it can be calculated from observable quantities, and requires no knowledge of the details of the ion-channel mechanism or its rate constants. The accuracy of this approximation is tested by exact calculations of the properties of runs of single openings, and of single bursts, for two specific mechanisms and a large range of rate constants. The approximation is good in all cases in which openings occur singly, or in closely spaced bursts. If, as is common in practice, openings occur in clusters that are separated by long shut periods, then overlap of clusters from two different channels may be detected, if no double opening is produced, as a period in the middle of a cluster in which the probability of being open doubles. The results derived here can be applied to such a period to test whether it results from the simultaneous activity of two channels, rather than from a change in the properties of a single channel.     

Study of fractal properties of single ionic channel gating mechanism by the fast Fourier transform]

Sets of the channel open times (tau o), closed times (tau c) and the full set of the channel open and closed times (tau o, tau c) in the activity of single Ca(2+)-activated K+ channels in cultured kidney cells Vero were analyzed using the fast Fourier transform. It was found that in the low-frequency range (about 0.01-10 Hz), power density can be described by the equation S(f) approximately f-alpha (as a rule, 0 < alpha < 1), and this part of the Fourier spectrum usually consists of narrow peaks at almost multiple frequencies. It was shown that the upper frequency boundary of this spectrum is determined by the kinetic parameters tau o [symbol: see text] tau c. The data obtained show that ion channel gating is a fractal process (correlated in time) and can be regarded as a random signal modulated by some periodical functions (sinuses). The data obtained by the Fourier method are in agreement with the earlier results obtained using the rescaled-range analysis.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Singular Value Decomposition of the Radial Distribution Function for Hard Sphere and Square Well Potentials

We compute the singular value decomposition of the radial distribution function for hard sphere, and square well solutions. We find that decomposes into a small set of basis vectors allowing for an extremely accurate representation at all interpolated densities and potential strengths. In addition, we find that the coefficient vectors describing the magnitude of each basis vector are well described by a low-order polynomial. We provide a program to calculate in this compact representation for the investigated parameter range.     

Statistical analysis of channel current from a membrane patch. II. A stochastic theory of a multi-channel system in the steady-state

A general stochastic theory is presented for analysis of current records of a patch containing an arbitrary number (N) of independent homologous channels in the steady-state. We give the "basic theorem" that at the instant of any open (or shut) transition of a channel, the other N-1 channels are located in each state with a probability equal to those in the steady-state, if enough transitions are observed. Using the "basic theorem", we derived: (a) the time-dependent open and shut frequencies after a definite type of transition, and (b) the probability density functions (pdf) of the duration of any period between two successive transitions. Briefly, the main results obtained were: (1) The time-dependent open (or shut) transition frequency after every shut (or open) transition at t = 0 in an N-channel patch, fJSh,Op(t)(N) (or fJOp,Sh(t)(N)), is the same as that of a one-channel patch except for the value of the constant. (2) In the all-shut (or all-open) period of a patch, the average duration of the period is 1/N, and the slowest exponential decay constant contained in the pdf is N times those of a single channel patch, respectively. (3) An example calculation for small N showed that the stochastic properties of a single channel can be obtained even when N is uncertain, if the channel open probability is small and exponential decay constants are separated. (4) When the channels are in equilibrium, the pdf of duration of every type of period in the patch is described by a sum of exponential terms with positive coefficients. This also holds for fJSh,Op(t)(N) and fJOp,Sh(t)(N).     

Cholinergic excitation of smooth muscles: Multiple signaling pathways linking M2 and M3 muscarinic receptors to cationic channels

Acetylcholine, the main neurotransmitter of the parasympathetic nervous system, depolarizes various smooth muscles and initiates their contraction via activating muscarinic cholinergic receptors. In most visceral smooth muscle tissues, such as the gastrointestinal tract, airways, and the urinary system, muscarinic receptors are comprised of predominant M₂ (about 80%)and minor M₃ (about 20%) subtypes. Cholinergic excitation is generally mediated by the opening of ion channels selective for monovalent cations (under physiological conditions, Na⁺ and K⁺); among them the cationic channel of an about 60 pS unitary conductance has been recently identified as the main target for acetylcholine action. The signal transduction leading to channel opening is very complex and involves activation of G_o protein (an M₂ effect), activation of phospholipase C (an M₃ effect), and [Ca²⁺]_i and voltage dependence of channel opening. These multiple signaling pathways were difficult to reconcile with the channel gating mechanisms since only a simplified two-state channel mechanism (e.g., one open and one shut state) was until recently available. However, our recent studies of channel gating in isolated outside-out membrane patches revealed a greater complexity. Thus, this cationic channel shows transitions between at least eight states, four open and four shut, with strong connections between adjacent shut and open states. Therefore, four pairs of connected states have been identified, which showed voltage-dependent transitions in each pair of shut/open states. Since the membrane potential did not affect the relative proportions between the pairs, we have assumed that these effects are controlled by ligands that bind to the channel and, thus, stabilize its various open conformations. In this work, direct tests of the above hypothesis have been performed, and their results showed that spontaneous brief channel gating exists in the absence of receptor or G-protein activation, which is strongly voltage-dependent (increasing at depolarized potentials). Furthermore, this activity was potentiated at a low agonist concentration, while channel openings generally remained brief. An increasing receptor occupancy by the agonist produced long channel openings, indicating a shift of gating towards a long open/brief shut pair of the channel states. These findings are interpreted in the context of the established signal transduction pathways;certain predictions for the whole-cell current are also examined.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 446–454, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

On the stochastic properties of single ion channels

It is desirable to be able to predict, from a specified mechanism, the appearance of currents that flow through single ion channels (a) to enable interpretation of experiments in which single channel currents are observed, and (b) to allow physical meaning to be attached to the results observed in kinetic (noise and relaxation) experiments in which the aggregate of many single channel currents is observed. With this object, distributions (and the means) are derived for the length of the sojourn in any specified subset of states (e.g. all shut states). In general these are found to depend not only on the state in which the sojourn starts, but also on the state that immediately follows the sojourn. The methods described allow derivation of the distribution of, for example, (a) the number of openings, and total length of the burst of openings, that may occur during a single occupancy, and (b) the apparent gap between such bursts. The methods are illustrated by their application to two simple theories of agonist action. The Castillo-Katz (non-cooperative) mechanism predicts, for example, that the number of openings per occupancy, and the apparent burst length, are independent of agonist concentration whereas a simple cooperative mechanism predicts that both will increase with agonist concentration.     

Kinetics of 9-aminoacridine block of single Na channels

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.     

Monotonic and non-monotonic single channel open time distributions with two sequential open states

Some conditions under which kinetic schemes including two sequential open states of identical conductance will display a non-monotonic (i.e. with a deficit of short open times and a maximum at t>0) distribution of single channel open times are described theoretically. Neither a closed cyclic scheme nor exclusively irreversible transitions between states are required for non-monotonic distributions. A required condition for the schemes considered here is that all openings are to a state from which closing is not possible. It is the presence of a precursor process to channel closing that produces the non-monotonic distribution. Following each channel opening some time is required for a transition into the second open state from which all closings proceed. Simple schemes of this sort cannot provide the basis of any experimental reports of non-monotonic distributions.     

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17-21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 microM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Permeable ions differentially affect gating kinetics and unitary conductance of L-type calcium channels

Although ion permeation and gating of L-type Ca(2+) channels are generally considered separate processes controlled by distinct components of the channel protein, ion selectivity can vary with the kinetic state. To test this possibility, we studied single-channel currents (cell-attached) of recombinant L-type channels (Ca(V)1.2, beta(2a), and alpha(2)delta) transiently expressed in tsA201 cells in the presence of the channel agonist BayK 8644 which promotes long channel openings (Mode 2 openings). We found that both the brief (Mode 1) and long (Mode 2) mean open times in the presence of Ca(2+) were relatively longer than those with Ba(2+). The unitary slope conductance with Ba(2+) was significantly larger (p<0.05) in Mode 2 openings than for brief Mode 1 openings, whereas the conductance with Ca(2+) did not vary with mode gating. Consequently, the gamma(Ba):gamma(Ca) ratio was greater for Mode 2 than Mode 1 openings. Our findings indicate that both ion permeation and gating kinetics of the L-type channel are differentially modulated by permeable ions. Ca(2+) binding to the L-type channel may stabilize the alteration of channel ion permeability mediated by gating kinetics, and thus, play a role in preventing excessive ion entry when the activation gating of the channel is promoted to the prolonged open state.     

Inactivation viewed through single sodium channels

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.     

Fractal properies of gating in potential-dependent K+-channels in Lymnaea stagnalis neurons

Sets of the channel open times, [tau(o)], and closed times, [tau(c)], and the full set of the channel open and closed times, [tau(o), tau(c)], in the activity of single voltage-dependent K+-channels in mollusc L. stagnalis neurons were analyzed using the rescaled range analysis (Hurst method), fast Fourier and wavelet transforms. It was found that the Hurst dependence for each time series could be approximated by a polygonal line with at least two slopes: H1 and H2 (Hurst exponents). The averaged values of H1 and H2 for the sets [tau(o), tau(c)] were equal to 0.61 +/- 0.03 and 0.83 +/- 0.11, respectively; for the [tau(o)] sets H1 = 0.66 +/- 0.03 and H2 = 0.95 +/- 0.10; for the [tau(c)] sets, H1 = 0.62 +/- 0.05 and H2 = 0.85 +/- 0.10. In some cases, a third slope appeared on the Hurst dependences. It was very variable and ranged between 0.5 and 1. The Hurst exponents H1, H2, and H3 characterized short, intermediate, and long time ranges, respectively. The ranges greatly varied from experiment to experiment. The data obtained show that the channel openings and closings (gating process) represent a persistent process correlated in time. The randomization of the time sets resulted in a single slope, H, of 0.52 +/- 0.02 characteristic of random processes. The results were confirmed by the fast Fourier and wavelet transforms. In addition, possible voltage dependences of Hurst exponents and their correlation with tau(o) and tau(c) were investigated. As a whole, single channel activity may be characterized as a multifractal process with a slight voltage dependence of the Hurst exponents.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

An extrapolation method for reducing equilibration times in sedimentation equilibrium experiments.

We present a detailed investigation of the use of an extrapolation technique to decrease running times of sedimentation equilibrium experiments. If concentration profiles are available at time delta tau, 2delta tau, 3delta tau,...., cn(r) = c(r, n delta tau), then the Aitken transformation replaces the cn(r) + ĉn(r) = [cn + 1(r) cn - 1(r) - c2n(r)]/[cn + 1(r) + cn - 1(r) - 2cn(r)]. We show that the ĉn(r) converge to the equilibrium values c infinity (r) much more quickly than the cn(r). Savings in time are shown to range from a factor of approximately 2 for meniscus depletion experiments to factors of between 4 and 8 for lower speeds or smaller molecular weights. It is also shown that the technique is quite sensitive to noise, so that an accurate optical system is required to allow its optimal use.