Baroreceptor mechanisms at the cellular level

Nothing is known of transduction mechanisms of baroreceptors in vivo. Not even the site of transduction is known. However, there are mechanotransducer ion channels that provide a useful model system of transduction. In these channels, transduction is accomplished by a strain-dependent increase in the probability of being open. Membrane tension is coupled to the channel by cytoskeletal strands that concentrate the strain energy from a large (approximately equal to 4000 A diameter) area of membrane and thereby provide high sensitivity. The channel is fast and does not inactivate, but viscoelastic coupling to the channel can dramatically alter the transfer function.     

Transduction of membrane tension by the ion channel alamethicin.

Mechanoelectrical transduction in biological cells is generally attributed to tension-sensitive ion channels, but their mechanisms and physiology remain controversial due to the elusiveness of the channel proteins and potential cytoskeletal interactions. Our discovery of membrane tension sensitivity in ion channels formed by the protein alamethicin reconstituted into pure lipid membranes has demonstrated two simple physical mechanisms of cytoskeleton-independent transduction. Single channel analysis has shown that membrane tension energizes mechanical work for changes of conductance state equal to tension times the associated increase in membrane area. Results show a approximately 40 A2 increase in pore area and transfer of an 80-A2 polypeptide into the membrane. Both mechanisms may be implicated in mechanical signal transduction by cells.     

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.     

Acetylcholine receptor: channel-opening kinetics evaluated by rapid chemical kinetic and single-channel current measurements.

A combination of rapid chemical kinetic (quench-flow) and single-channel current measurements was used to evaluate kinetic parameters governing the opening of acetylcholine-receptor channels in the electric organ (electroplax) of Electrophorus electricus. Chemical kinetic measurements made on membrane vesicles, prepared from the E. electricus electroplax, using carbamoylcholine (200 microM-20 mM) at 12 degrees C, pH 7.0, and in the absence of a transmembrane voltage, yielded values for K1 (dissociation constant for receptor activation), phi (channel closing equilibrium constant), J (specific reaction rate for ion flux), and alpha max (maximum inactivation rate constant) of 1 mM, 3.4, 4 x 10(7) M-1 s-1, and 12 s-1, respectively. The single-channel current recordings were made with cells also from the E. electricus electroplax, at the same temperature and pH as the chemical kinetic measurements, using carbamoylcholine (50 microM-2 mM), acetylcholine (500 nM), or suberyldicholine (20 nM). Single-channel current measurements indicated the presence of a single, unique open-channel state of the E. electricus receptor, in concurrence with previous, less extensive measurements. The rate constant for channel closing (kc) obtained from the mean open time of the receptor channel is 1,100 s-1 for carbamoylcholine, 1,200 s-1 for acetylcholine, and 360 s-1 for suberyldicholine at zero membrane potential; and it decreases e-fold for an 80 mV decrease in transmembrane voltage in each case. The decrease in mean open times of the receptor channel that is associated with increasing the carbamoylcholine concentration is interpreted to be due to carbamoylcholine binding to the regulatory (inhibitory) site on the receptor. An analysis of data obtained with carbamoylcholine showed that the closed times within a burst of channel activity fit a two-exponential distribution, with a concentration-independent time constant considered to be the time constant for carbamoylcholine to dissociate from the regulatory site, and a carbamoylcholine concentration-dependent, but voltage-independent, time constant interpreted to represent the rate constant for channel opening (k0). An analysis of the mean closed time data on the basis of the minimum model gives values for K1 and k0 of 0.6 mM and 440 s-1, respectively, with carbamoylcholine as the activating ligand. The values obtained for K1, phi (= kc/k0), J, and alpha from the single-channel current measurements using electroplax are in good agreement with the values obtained from the chemical kinetic measurements using receptor-rich vesicles prepared from the same cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ageing on the expression of protein kinase C and its activation by 1,25(OH)2-vitamin D3 in rat skeletal muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH)2-vitamin D3 [1,25(OH)2D3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms alpha and beta, novel isoforms delta, epsilon, and theta and atypical isoform zeta were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH)2D3. The hormone (10(-9) M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC alpha, beta and delta was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms epsilon, theta and zeta. After a short exposure (1 min) of muscle to 1,25(OH)2D3, increased amounts of PKC alpha and beta in muscle membranes and reverse translocation (from membrane to cytosol) of PKC epsilon were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (alpha and beta) and calcium-independent PKC (delta, epsilon, theta and zeta) signal transduction pathways under selective regulation by 1,25(OH)2D3.     

Force Transduction and Lipid Binding in MscL: A Continuum-Molecular Approach

The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, and identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin.     

Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.     

Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium.

The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca(2+) regulation in cardiac muscle. Pulse photolysis of caged P(i) (alpha-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k (rho i), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M(-1) s(-1), which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca(2+) activation to 20% of maximum tension reduced k (rho i) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca2+. A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca(2+)-sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca(2+), suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca(2+). The apparent rate constant, k(Ca), was accelerated in the presence of high P(i) consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca(2+) activation. However, significant quantitative differences between k (rho i) and k(Ca), including different sensitivities to Ca(2+) and P(i) indicate that caged Ca(2+) tension transients are influenced by additional Ca(2+)-dependent but P i-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca(2+) regulatory system and partially limit the physiological rate of tension development in cardiac muscle.     

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.     

Ventilation is unstable during drowsiness before sleep onset.

Ventilation is unstable during drowsiness before sleep onset. We have studied the effects of transitory changes in cerebral state during drowsiness on breath duration and lung volume in eight healthy subjects in the absence of changes in airway resistance and fluctuations of ventilation and CO2 tension, characteristic of the onset of non-rapid eye movement sleep. A volume-cycled ventilator in the assist control mode was used to maintain CO2 tension close to that when awake. Changes in cerebral state were determined by the EEG on a breath-by-breath basis and classified as alpha or theta breaths. Breath duration and the pause in gas flow between the end of expiratory airflow and the next breath were computed for two alpha breaths which preceded a theta breath and for the theta breath itself. The group mean (SD) results for this alpha-to-theta transition was associated with a prolongation in breath duration from 5.2 (SD 1.3) to 13.0 s (SD 2.1) and expiratory pause from 0.7 (SD 0.4) to 7.5 s (SD 2.2). Because the changes in arterial CO2 tension (PaCO2) are unknown during the theta breaths, we made in two subjects a continuous record of PaCO2 in the radial artery. PaCO2 remained constant from the alpha breaths through to the expiratory period of the theta breath by which time the duration of breath was already prolonged, representing an immediate and altered ventilatory response to the prevailing PaCO2. In the eight subjects, the CO2 tension awake was 39.6 Torr (SD 2.3) and on assisted ventilation 38.0 Torr (1.4). We conclude that the ventilatory instability recorded in the present experiments is due to the apneic threshold for CO2 being at or just below that when awake.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.     

Membrane stretch affects gating modes of a skeletal muscle sodium channel.

The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension.     

Kinetic characteristics of cross bridges in the heart muscles by a temperature jump method]

Experiments were performed on rat skinned trabecular muscles dissected from the right ventricles. They were activated (pCa = 5.9) at 10 C, 2.3 nm sarcomere length. The temperature jump induced the biexponential tension rise. The rate constant of the fast tension rise (k1) was 3-4.5 ms and that of the slow tension rise (k2) was about 15 ms. These values were slower (3 times) comparing with those of a single skeletal fibre. The discrepancy can be explained by different kinetic properties of heart and skeletal myosin or presents a well developed connection tissue (series elastic element) in the heart muscle.     

Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers

Lipid metabolites, free fatty acids and lysophospholipids, modify the function of membrane proteins including ion channels. Such alterations can occur through signal transduction pathways, but may also result from "direct" effects of the metabolite on the protein. To investigate possible mechanisms for such direct effects, we examined the alterations of gramicidin channel function by lysophospholipids (LPLs): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The experiments were done on planar bilayers formed by diphytanoylphosphatidylcholine in n-decane a system where receptor- mediated effects can be excluded. At aqueous concentrations below the critical micelle concentration (CMC), LPLs can increase the dimerization constant for membrane-bound gramicidin up to 500-fold (at 2 microM). The relative potency increases as a function of the size of the polar head group, but does not seem to vary as a function of head group charge. The increased dimerization constant results primarily from an increase in the rate constant for channel formation, which can increase more than 100-fold (in the presence of LPC and LPI), whereas the channel dissociation rate constant decreases only about fivefold. The LPL effect cannot be ascribed to an increased membrane fluidity, which would give rise to an increased channel dissociation rate constant. The ability of LPC to decrease the channel dissociation rate constant varies as a function of channel length (which is always less than the membrane's equilibrium thickness): as the channel length is decreased, the potency of LPC is increased. LPC has no effect on membrane thickness or the surface tension of monolayers at the air/electrolyte interface. The bilayer-forming glycerolmonooleate does not decrease the channel dissociation rate constant. These results show that LPLs alter gramicidin channel function by altering the membrane deformation energy, and that the changes in deformation energy can be related to the molecular "shape" of the membrane-modifying compounds. Similar alterations in the mechanical properties of biological membranes may form a general mechanism by which one can alter membrane protein function.     

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

A virtual hair cell, II: evaluation of mechanoelectric transduction parameters

The virtual hair cell we have proposed utilizes a set of parameters related to its mechanoelectric transduction. In this work, we observed the effect of such channel gating parameters as the gating threshold, critical tension, resting tension, and Ca(2+) concentration. The gating threshold is the difference between the resting and channel opening tension exerted by the tip link assembly on the channel. The critical tension is the tension in the tip link assembly over which the channel cannot close despite Ca(2+) binding. Our results show that 1), the gating threshold dominated the initial sensitivity of the hair cell; 2), the critical tension minimally affects the peak response, (I), but considerably affects the time course of response, I(t), and the force-displacement, F-X, relationship; and 3), higher intracellular [Ca(2+)] resulted in a smaller fast adaptation time constant. Based on the simulation results we suggest a role of the resting tension: to help overcome the viscous drag of the hair bundle during the oscillatory movement of the bundle. Also we observed the three-dimensional bundle effect on the hair cell response by varying the number of cilia forced. These varying forcing conditions affected the hair cell response.     

Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy

The three-dimensional structure of the skeletal muscle voltage-gated L-type calcium channel (Ca(v)1.1; dihydropyridine receptor, DHPR) was determined using electron cryo-microscopy and single-particle averaging. The structure shows a single channel complex with an approximate total molecular mass of 550 kDa, corresponding to the five known subunits of the DHPR, and bound detergent and lipid. Features visible in our structure together with antibody labeling of the beta and alpha(2) subunits allowed us to assign locations for four of the five subunits within the structure. The most striking feature of the structure is the extra-cellular alpha(2) subunit that protrudes from the membrane domain in close proximity to the alpha(1) subunit. The cytosolic beta subunit is located close to the membrane and adjacent to subunits alpha(1), gamma and delta. Our structure correlates well with the functional and biochemical data available for this channel and suggests a three-dimensional model for the excitation-contraction coupling complex consisting of DHPR tetrads and the calcium release channel.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.