K(+)-channel blockers do not decrease acetylcholine depolarizations in canine trachealis.

Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10(-6) M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20-30 mV positive to the resting potential, previously shown to be approximately -55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10(-6) M), charybdotoxin (10(-7) M), and glybenclamide (10(-5) M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10(-6) M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K(+)-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K(+)-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.     

A slow voltage-activated potassium current in rat vagal neurons.

Potassium currents play a key role in controlling the excitability of neurons. In this paper we describe the properties of a novel voltage-activated potassium current in neurons of the rat dorsal motor nucleus of the vagus (DMV). Intracellular recordings were made from DMV neurons in transverse slices of the medulla. Under voltage clamp, depolarization of these neurons from hyperpolarized membrane potentials (more negative than -80 mV) activated two transient outward currents. One had fast kinetics and had properties similar to A-currents. The other current had an activation threshold of around -95 mV (from a holding potential -110 mV) and inactivated with a time constant of about 3s. It had a reversal potential close to the potassium equilibrium potential. This current was not calcium dependent and was not blocked by 4-aminopyridine (5 mM), catechol (5 mM) or tetraethylammonium (20 mM). It was completely inactivated at the resting membrane potential. This current therefore represents a new type of voltage-activated potassium current. It is suggested that this current might act as a brake to repetitive firing when the neuron is depolarized from membrane potentials negative to the resting potential.     

Calcium-dependence of Donnan potentials in glycerinated rabbit psoas muscle in rigor, at and beyond filament overlap; a role for titin in the contractile process

In glycerinated rabbit psoas muscle, Donnan potential measurements demonstrated that the net electric charge on the actin-myosin matrix undergoes a sharp switch-like transition at pCa₅₀ = 6.8. The potentials are 2 mV less negative at the lower pCa²⁺ (P < 0.001). If ATP is present, the muscle contracts and breaks the microelectrode. Therefore the rigor state was studied. There is no reason to suppose a priori that a similar voltage switch does not occur during contraction, however.Calcium dependence is still apparent in muscles stretched beyond overlap (sarcomere length > 3.8 μm) and is also seen in the gap filaments between the A- and I-band ends; further stretching abolishes the dependence. These experiments strongly suggest that calcium dependence is controlled initially by the titin component, and that this control is lost when titin filaments break. We suppose that that effect is mediated by the titin kinase in the M-line region and may involve the extensible PEVK region of titin.There is great interest in the electric charge on proteins in muscle within the structural system. We suggest how changes in these charges may control the calcium activation process. We also suggest some simple experimental approaches that could clarify these effects.     

The role of the membrane potential in chondrocyte volume regulation

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium‐selective and chloride‐selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium‐sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔV_m = ?30 ± 4 mV). Analysis of the gadolinium‐sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔV_m = ?18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at ?80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979–2986, 2011. © 2011 Wiley‐Liss, Inc.     

Voltage-sensitive calcium flux promoted by vesicles in an isolated cardiac sarcolemma preparation

Summary The effect of membrane potential on the vesicular uptake of calcium in an isolated cardiac sarcolemma preparation from canine ventricle was evaluated. Membrane potentials were developed by the establishment of potassium gradients across the vesicular membranes. In the presence of valinomycin, the fluorescence changes of the voltage sensitive dye, diS-C₃-(5) were consistent with the development of potassium equilibrium potentials. Using EGTA to remove endogenous calcium from the preparation and to maintain a low intravesicular calcium concentration over time, the uptake of calcium was linear from 5 to 100 sec, in the absence of sodium, at both –98 and –1 mV. The rate of calcium uptake (calcium influx) was approximately twofold greater at –1 mV than at –98 mV, and prepolarization of the membrane potential to –98 mV did not enhance calcium influx upon subsequent depolarization to –1 mV. Hence, calcium influx was voltage-sensitive but not depolarization-induced and did not inactivate with time. Furthermore, the calcium influx was not inhibited by the organic calcium antagonists, which suggests that this flux did not occur via the transient calcium channel. Evaluation of calcium influx over a wide range of membrane potentials produced a profile consistent with the hypothesis that calcium entered the vesicles through the pathway responsible for the persistent inward current observed in voltage-clamped isolated myocytes. A model was proposed to account for these results.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina

Electrical activity in the fertilized egg of the tunicate Clavelina was studied with microelectrode recording and voltage clamp techniques. The resting potential could assume either of two stable values (approximately ?70 or ?30 mV) and could be shifted between these values by direct current stimulation. Spontaneous shifts between two stable resting potentials were also seen. Egg cells produced action potentials spontaneously and in response to depolarizing stimuli. Inward currents were carried by both Na and Ca ions and a prominent outward potassium current was seen with depolarization to voltages above ?15 mV. The steady-state current-voltage relationship (I–V curve) of the membrane showed two voltages where the net membrane current equaled zero: approximately ?35 and ?70 mV. Between these two voltages, membrane current was inward and carried by noninactivating Na and Ca currents. Inward rectification, which was blocked by external Rb, occurred at voltages below ?70 mV. The voltage dependence of inward rectification is thought by the authors to be important for establishing the more negative resting potential; it is also thought the presence of inward current which does not inactivate completely at voltages more negative than about ?20 mV is an important determinant of the more depolarized resting potential.     

Membrane permeabilities determining resting,action and mechanoreceptor potentials inStentor coeruleus

Summary In response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl₂ concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation whereP _Ca/P _K = 0.068 andP _Cl/P _K = 0.072. The intracellular ionic activities (K _i ⁺ = 13.1 mM, Cl _i ^– = 9.9 mM, Ca _i ⁺ = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (K_i=12.4 mM, Cl_i = 9.4 mM) except in the case of Ca_i where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl₂ concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl^– concentration. These peak potentials are well described by assuming that theP _Ca/P _k = 7.9 andP _Cl/P _K=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl₂ concentration in a manner similar to that of the action potential peak. As in the case of the action potential both theP _Ca/P _k andp _cl/p _k ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K⁺, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.     

Two excitatory motoneurons differ in quantal content of their junctional potentials in abdominal muscle fibers of the cricket,Gryllus bimaculatus

In abdominal muscles 202 and 203 of the cricket, Gryllus bimaculatus, large and small excitatory junctional potentials (l- and s-EJPs) with similar durations can be recorded from the same muscle fibers. At the normal extracellular calcium ion concentration ([Ca(2+)](o)) of 5mM, the amplitudes of l-EJPs in both muscles were larger than the threshold membrane potential for muscle action potentials, which is about -40mV. Below 0.75mM [Ca(2+)](o), the amplitudes became much smaller and were below the firing level for the action potentials. At 0.5mM, they fluctuated and decreased to 10.3 and 1.9mV in muscles 202 and 203, respectively, and at 0.25mM frequent failures occurred. The amplitudes of s-EJPs at 5mM [Ca(2+)](o) were 13.3 and 5.1mV in muscles 202 and 203, respectively, and the fluctuating amplitudes were far below the threshold for muscle action potentials. Below 0.75mM, s-EJPs were rarely observed. The relation between log(EJP amplitude) and log([Ca(2+)](o)) was linear within a certain range of [Ca(2+)](o) and the slopes of the lines for l-EJPs were about twice as steep as those for s-EJPs in both muscles. In muscle 202, the amplitude distribution of l-EJPs obtained at 0.25mM and that of s-EJPs at 0.75mM both showed peaks at once and twice the voltage at the first peak, which were coincident with the voltages at the peaks of amplitude distributions of miniature EJPs recorded simultaneously. The reversal potentials for l- and s-EJPs in muscle 202 were +1.02 and +0.22mV, respectively. In muscle 202, the decreases in amplitude of both EJPs by L-glutamate were similar and concentration-dependent. The results suggest that the difference in amplitude between l- and s-EJPs is attributable mainly to the difference in quantal contents.     

Models of active transport of neurotransmitters in synaptic vesicles

Models of the active transport of neurotransmitters in synaptic vesicles were constructed. The models were used to determine the resting potential at membranes of synaptic vesicles: 40mV (monoamines and acetylcholine) and -40mV (glutamate). The potential at the membrane of a synaptic vesicle was almost absent for the transport of GABA and glycine. The neurotransmitter concentration of a cell was 0.1-18mM at the concentration of neurotransmitters in a vesicle equal to 0.5M. This result is in qualitative agreement with the relevant experimental data.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Effects of temperature on properties of flight neurons in the locust

High ambient temperatures increase the wing-beat frequency in flying locusts, Locusta migratoria. We investigated parameters of circuit and cellular properties of flight motoneurons at temperatures permissive for flight (20–40 °C). As the thoracic temperature increased motoneuronal conduction velocity increased from an average of 4.40 m/s at 25 °C to 6.73 m/s at 35 °C, and the membrane time constant decreased from 11.45 ms to 7.52 ms. These property changes may increase locust wing-beat frequency by affecting the temporal summation of inputs to flight neurons in the central circuitry. Increases in thoracic temperature from 25–35 °C also resulted in a hyperpolarization of the resting membrane potentials of flight motoneurons from an average of-41.1 mV to -47.5 mV, and a decrease of input resistances from an average of 3.45 M to 2.00 M. Temperature affected the measured input resistance both by affecting membrane properties, and by altering synaptic input. We suggest that the increase in conduction velocity Q₁₀=1.53) and the decrease of membrane time constant (Q₁₀=0.62) would more than account for the wing-beat frequency increase (Q₁₀=1.15). Hyperpolarization of the resting membrane potential (Q₁₀=1.18) and reduction in input resistance (Q₁₀=0.54) may be involved in automatic compensation of temperature effects.Abbreviations ANOVA analysis of variance - CPG central pattern generator - DL dorsal longitudinal muscles - EMG electromyographic - MN motoneuron - PSP post synaptic potential - Q₁₀ temperature coefficient - RMP resting membrane potential - S.D. standard deviation - SR stretch receptor     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba²⁺ currents through voltage gated Ca²⁺ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca²⁺ channel blockers, volatile anesthetics blocked Ca²⁺ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca²⁺ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca²⁺ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.     

Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O₂. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 μM NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV ± 3.1 (n = 4) and +40 ± 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl₂ (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.     

Determination of ionic calcium in frog skeletal muscle fibers

Ionic calcium concentrations were measured in frog skeletal muscle fibers using Ca-selective microelectrodes. In fibers with resting membrane potentials more negative than -85 mV, the mean pCa value was 6.94 (0.12 microM). In fibers depolarized to -73 mV with 10-mM K the mean pCa was 6.43 (0.37 microM). This increase in the intracellular [Ca2+] could be related to the higher oxygen consumption and heat production (Solandt effect) reported to occur under these conditions. Caffeine, 3 mM, also produced an increase in the free ionic calcium to a pCa of 6.52 (0.31 microM) without changes in the membrane potential. Lower caffeine concentrations, 1 and 2 mM, did not change the fiber pCa. Lower Ca concentrations in the external medium effectively reduced the internal ionic calcium to an estimated pCa of 7.43 (0.03 microM).     

The combined effect of Cd2+ and ACh on action potentials of Nitellopsis obtusa cells

Interrelations between the action of acetylcholine (ACh) and cadmium ions (Cd²⁺) on bioelectrogenesis of Nitellopsis obtusa cells were investigated. We analyzed repetitively triggered action potentials (AP), their reproducibility, shape and dynamics of membrane potential after AP induction. ACh significantly increased membrane permeability only at high concentrations (1 mM and 5 mM). Repolarisation level of action potential after the first stimulus was much more positive in all cells treated with ACh as compared to the control. Differences of membrane potentials between points just before the first and the second stimuli were 23.4±.0 mV (control); 40.4±5.9 mV (1 mM ACh solution) and 57.7 ± 8.5 mV (5 mM ACh solution). Cd²⁺ at 20 μM concentration was examined as a possible inhibitor of acetylcholinesterase (AChE) in vivo. We found that cadmium strengthens depolarizing effect of acetylcholine after the first stimulus. The highest velocity of AP repolarization was reduced after ACh application and Cd²⁺strengthened this effect. There were no differences in dynamics of membrane potential after repetitively triggered action potentials in ACh or ACh and Cd²⁺ solutions. This shows that cadmium in small concentration acts as inhibitor of acetylcholinesterase.     

Ion Levels and Membrane Potential in Chick Heart Tissue and Cultured Cells

Intracellular concentrations of sodium and potassium as well as resting potentials and overshoots have been determined in heart tissue from chick embryos aged 2–18 days. Intracellular potassium declined from 167 mM at day 2 to 117–119 mM at days 14–18. Intracellular sodium remained nearly constant at 30–35 mM during the same period. The mean resting potential increased from -61.8 mV at day 3 to about -80 mV at days 14–18. The mean overshoot during the same period increased from 12 to 30 mV. P_Na/P_K calculated from the ion data and resting potentials declined from 0.08 at day 3 to 0.01 at days 14–18. Thus, the development of embryonic chick heart during days 2–14 is characterized by a declining intracellular potassium concentration and an increasing resting potential and overshoot. Heart cells from 7- to 8-day embryos, cultured either in monolayer or reassociated into aggregates, were compared with intact tissue of the same age. The intracellular concentrations of sodium and potassium were similar in the three preparations and cultured cells responded to incubation in low potassium medium or treatment with ouabain in a manner similar to that of intact tissue. Resting potentials and overshoots were also similar in the three preparations.     

A packed Cytodex microbead array for three-dimensional cell-based biosensing

A packed Cytodex 3 microbead array was fabricated as a simple three-dimensional (3-D) cell-based biosensing format. Resting membrane potentials and voltage-gated calcium channel (VGCC) function of SH-SY5Y human neuroblastoma cells cultured on the microbead array versus collagen-coated flat (2-D) substrates were evaluated by confocal microscopy with a potentiometric dye, tetramethylrhodamine methyl ester, and a calcium fluorescent indicator, Calcium Green-1. SH-SY5Y cells, differentiated with 1mM dibutyryl cAMP and 2.5 microM 5-bromodeoxyuridine, showed significant resting membrane potential establishment on the topographical scaffolds in a period of 13 days into differentiation, in contrast to the previously reported insignificant resting membrane potential establishment of the same cells within collagen hydrogels. On days 2, 8 and 13 into differentiation, cells on collagen-coated flat substrates developed resting membrane potentials of -6.0+/-19.5 mV (n=198), -30.5+/-19.9 mV (n=191) and -21.7+/-18.9 mV (n=308), in contrast to values for cells on 3-D scaffolds of -25.8+/-14.7 mV (n=112), -37.6+/-13.1 mV (n=120) and -28.7+/-12.2 mV (n=158), respectively. The development of VGCC function, as measured by percentage of cells responsive to 50 mM high K(+) depolarization, was significantly slower for cells on 3-D scaffolds (20.0% on day 13 into differentiation) than for cells on 2-D substrates (30.7% on day 8 into differentiation). The exaggerated 2-D cell calcium dynamics, in comparison with those of 3-D cells, is consistent with previous 2-D/3-D comparative studies. This study established the rationale and feasibility of the microbead array format for 3-D cell-based biosensing.