Superposition properties of independent ion channels

Membrane patches usually contain several ion channels of a given type. However, most of the stochastic modelling on which data analysis (in particular, estimation of kinetic constants) is currently based, relates to a single channel rather than to multiple channels. Attempts to circumvent this problem experimentally by recording under conditions where channel activity is low are restrictive and can introduce bias; moreover, possibly important information on how multichannel systems behave will be missed. We have extended existing theory to multichannel systems by applying results from point process theory to derive some distributional properties of the various types of sojourn time that occur when a given number of channels are open in a system containing a specified number of independent channels in equilibrium. Separate development of properties of a single channel and the superposition of several such independent channels simplifies the presentation of known results and extensions. To illustrate the general theory, particular attention is given to the types of sojourn time that occur in a two channel system; detailed expressions are presented for a selection of models, both Markov and non-Markov.     

Multiplexed on-column protein digestion and capillary electrophoresis for high-throughput comprehensive peptide mapping

A novel scheme based on multiplexed capillary electrophoresis (CE) has been developed for high-throughput, low-cost and comprehensive peptide mapping. Orthogonal peptide maps of the protein of interest were obtained by using multiple reaction conditions with three different enzymes (trypsin, pepsin, and chymotrypsin), and multiple separation conditions with six zone electrophoresis buffers and two micellar electrokinetic chromatography (MEKC) buffers. Fifteen nanoliters of two protein samples (beta-lactoglobulin A and beta-lactoglobulin B) were separately mixed on-column and digested independently at 37 degrees C for 10 min to produce peptides in a 20-capillary system. The resulting peptides were detected simultaneously at 214 nm by a photodiode array detector. The overall analysis time from reaction to detection was about 40 min.     

Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties

Endogenous serine proteases have been reported to control the reabsorption of Na(+) by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na(+) transport mediated by the epithelial Na(+) channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 +/- 10.5%) in the amiloride-sensitive sodium transport (I(Na)) with a time constant of 18 min and half maximal inhibition constant of 1 microM. Analysis of amiloride analogue blocker-induced fluctuations in I(Na) showed linear rate-concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947-C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of I(Na). A 50% decrease in I(Na) was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of I(Na) as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of I(Na) is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na(+) channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain I(Na) at some minimal threshold value.     

Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots

In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met₃SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met₃SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met₃SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met₃SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met₃SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.     

High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.     

A computer-controlled multichannel micropipetter

A multichannel micropipetter was developed capable of pipetting as little as 1 μl with a reproducibility of better than ±2% and an accuracy of ±0.5%. The micropipetter consists of a precision syringe to which 13 individually valved fluid channels are connected as a bundle of segments spreading out radially from the tip of the syringe to pinch valves and further to fluid interfaces consisting of steel tubing sections for uptake or dispensing of fluid. A stepping motor drives the piston of the measuring syringe by means of a precision screw. Motor and valves are under computer control. Low dead volume (internal volume, ca. 1 μl/channel; external volume, 0.3 μl/cm of tubing), and the absence of internal valving parts ensure low cross-conamination (ca. 0.1%). These features together with the versatility provided by the large number of independent channels and the automatic operation make the instrument suitable for pipetting multicomponent mixtures in the general biochemistry laboratory (for enzyme kinetics and complex reactions) as well as in specialized routine applications (clinical diagnostics and radioimmunoassay).     

Tracking acoustic transmitters by code division multiple access (CDMA)-based telemetry

This paper describes a new acoustic telemetry system based on the principles of code division multiple access (CDMA) whereby signal coding enables the simultaneous sub-meter monitoring of potentially hundreds of animals at high sampling rate (e.g. 5 s) and on a single frequency channel. The technology permits reliable operation under a variety of conditions including high ambient noise, multipath and in the presence of multiple tagged animals. The concepts of dilution of precision (DOP) and reliability index (RI) as performance measures of CDMA position solutions are introduced. DOP and RI are used in the design phase of a study to develop hydrophone array geometries that maximize position solution precision and minimize the occurrence of system-induced data outliers. Computed position solutions qualified by DOP and RI assist in data interpretation and provide an objective means of separating system induced outliers from animal behavior. To assess the suitability of CDMA telemetry for monitoring tagged animals under shallow water conditions, experiments were conducted to track the movement of blue crab (Callinectes sapidus) in a salt marsh pond. During the study, data from 32-tagged animals was collected by a CDMA telemetry system and used to provide preliminary information on individual animal movement. Performance results obtained from the study indicate that the precision of position solutions consistently achieved sub-meter levels under shallow water conditions (depth <0.5 m). Accumulated detection statistics indicate that the nominal probability of detection exceeded 80% at individual hydrophone channels. The ability to simultaneously monitor the tagged population of 32 animals at a sampling rate of 5 s was verified.     

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir‐glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi‐enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP‐CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi‐enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high‐throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP‐CE analysis is demonstrated for enzyme modules for the synthesis of UDP‐α‐D‐glucuronic acid (UDP‐GlcA) and UDP‐α‐D‐galactose (UDP‐Gal). In this way we achieve high space‐time‐yields: 1.8 g/L?h for UDP‐GlcA and 17 g/L?h for UDP‐Gal. The presented MP‐CE methodology has the impact to be used as general analytical tool for fast optimization of multi‐enzyme cascade reactions.     

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.     

Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots

The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met₃PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met₃PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met₃PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met₃PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met₃PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met₃PbCl binding site is located outside the channel selectivity filter.     

Time-Dependent Molecular Memory in Single Voltage-Gated Sodium Channel

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na⁺ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa_v1.2α channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na⁺ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a “pseudo-oscillatory” nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na⁺ channels. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na⁺ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.     

Statistical properties of ion channel records. Part II: estimation from the macroscopic current

Macroscopic ion channel current is the summation of the stochastic records of individual channel currents and therefore relates to their statistical properties. As a consequence of this relationship, it may be possible to derive certain statistical properties of single channel records or even generate some estimates of the records themselves from the macroscopic current when the direct measurement of single channel currents is not applicable. We present a procedure for generating the single channel records of an ion channel from its macroscopic current when the stochastic process of channel gating has the following two properties: (I) the open duration is independent of the time of opening event and has a single exponential probability density function (pdf), (II) all the channels have the same probability to open at time t. The application of this procedure is considered for cases where direct measurement of single channel records is difficult or impossible. First, the probability density function (pdf) of opening events, a statistical property of single channel records, is derived from the normalized macroscopic current and mean channel open duration. Second, it is shown that under the conditions (I) and (II), a non-stationary Markov model can represent the stochastic process of channel gating. Third, the non-stationary Markov model is calibrated using the results of the first step. The non-stationary formulation increases the model ability to generate a variety of different single channel records compared to common stationary Markov models. The model is then used to generate single channel records and to obtain other statistical properties of the records. Experimental single channel records of inactivating BK potassium channels are used to evaluate how accurately this procedure reconstructs measured single channel sweeps.     

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.     

A solid state signal multiplexer

A solid state device has been designed which is capable of scanning up to eight thermocouple inputs and feeding the output of each sequentially into a single channel recorder. Alternately, up to four thermocouples can be amplified and multiplexed with other analog signals to provide outputs compatible with a single span range. During normal operation this unit cycles repeatedly through a present number of channels and activates the recorder pen lift mechanism between channels. Subsequent circuitry features a unity gain differential input amplifier which can compensate all thermocouples from a single reference thermocouple, an adjustable gain amplifier, provision for unamplified uncompensated output from four channels, and provision for generating the first derivative of the thermocouple inputs to allow direct monitoring of either cooling rate or warming rate. Although designed to monitor cooling rates, the unit is sufficiently versatile to be useful for many other recording applications.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

Measuring kinetics of complex single ion channel data using mean-variance histograms.

The measurement of single ion channel kinetics is difficult when those channels exhibit subconductance events. When the kinetics are fast, and when the current magnitudes are small, as is the case for Na+, Ca2+, and some K+ channels, these difficulties can lead to serious errors in the estimation of channel kinetics. I present here a method, based on the construction and analysis of mean-variance histograms, that can overcome these problems. A mean-variance histogram is constructed by calculating the mean current and the current variance within a brief "window" (a set of N consecutive data samples) superimposed on the digitized raw channel data. Systematic movement of this window over the data produces large numbers of mean-variance pairs which can be assembled into a two-dimensional histogram. Defined current levels (open, closed, or sublevel) appear in such plots as low variance regions. The total number of events in such low variance regions is estimated by curve fitting and plotted as a function of window width. This function decreases with the same time constants as the original dwell time probability distribution for each of the regions. The method can therefore be used: 1) to present a qualitative summary of the single channel data from which the signal-to-noise ratio, open channel noise, steadiness of the baseline, and number of conductance levels can be quickly determined; 2) to quantify the dwell time distribution in each of the levels exhibited. In this paper I present the analysis of a Na+ channel recording that had a number of complexities. The signal-to-noise ratio was only about 8 for the main open state, open channel noise, and fast flickers to other states were present, as were a substantial number of subconductance states. "Standard" half-amplitude threshold analysis of these data produce open and closed time histograms that were well fitted by the sum of two exponentials, but with apparently erroneous time constants, whereas the mean-variance histogram technique provided a more credible analysis of the open, closed, and subconductance times for the patch. I also show that the method produces accurate results on simulated data in a wide variety of conditions, whereas the half-amplitude method, when applied to complex simulated data shows the same errors as were apparent in the real data. The utility and the limitations of this new method are discussed.     

Slow vacuolar channels of non-embryogenic and embryogenic cultures of winter wheat

Slowly activating vacuolar channels (SV), were examined in embryogenic and non-embryogenic cultures of winter wheat using a patch-clamp technique. Four different types of cultures were examined: embryogenic and non-embryogenic calli from embryos, embryogenic and non-embryogenic calli from inflorescences. In a cell-attached mode single SV channel events were recorded. Unitary conductance of single SV channels was between 37 pS and 48 pS and did not significantly depend on the kind of the culture, although it was a tendency that SV channels of embryogenic calli possessed lower unitary conductance than those of non-embryogenic. 2,4-D caused significant lowering of unitary conductance from 48±6 pS in the control culture of embryogenic embryos to 28±6 pS in vacuoles treated. The SV channel density was estimated as 0.34 μm⁻².     

Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase

Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.