Rapid Solution Changes Research Articles

Pharmacological characterization of a primary nicotinic receptor from the neuromuscular junction

Nicotinic receptors at the neuromuscular junction are important targets in the study of autoimmune disorders such as myasthenia gravis. Historic efforts targeting nAChR drug discovery focused on fluorescence based calcium flux assays along with low throughput manual patch clamp techniques due to its dependence on complete liquid exchange. With the advent of next generation automated patch champ instruments with integrated solution control and rapid solution change, extensive pharmacological characterization of the neuromuscular nAChR ((α1)2β1γδ) is now possible.

Increased Retention of Cardiac Cells to a Glass Substrate through Streptavidin-Biotin Affinity.

In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin–biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surface-engineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.

Smart Hydroponic Greenhouse (Sensing, Monitoring and Control) Prototype Based on Arduino and IOT

Aims: Sensing, monitoring and control the micro-climate measurements and environmental conditions of greenhouse prototype to create a smart hydroponic greenhouse for maximizing the food production as well as minimizing the ecological footprint under the climate change impacts, Coved 19 crisis, and natural resources shortages.
 Study Design: Factorial with 3 replicates.
 Place and Duration of Study: Central Laboratory for Agriculture Climate (CLAC), Agriculture Research Center, Egypt during 2020.
 Methodology: Two systems of hydroponic culture, nutrient film technique (NFT) and deep flow technique (DFT) that cultivated by lettuce plants were established under greenhouse (polycarbonate) prototype (0.8 * 1.2 * 0.6 m) designed with artificial grown light and cooling system. Based on Arduino Mega 2560 that programmed via the Arduino IDE program, different sensors and actuators were used to establishing the smart greenhouse. Internet of things (IoT) via Node MCU ESP 8266 that programmed to transmitted the data every 30 min. to the internet web google platform (Firebase) for presenting the real-time records and hosting the data. Vegetative characteristics; yield parameters and N, P, and K contents of lettuce plants were measured.
 Results: the smart greenhouse worked according to the programming of Arduino Mega and Node MCU with high efficiency. Google firebase platform displays the real-time records and hosts about 100 thousand different sensor records during the lettuce season. Decreasing the distance between the lettuce and artificial lightning source led to increasing the intensity of light that had a positive impact on lettuce growth but it wasn't sufficient to give a high quality of lettuce yield under the experiment. NFT system gave higher values of average No. of leaves and fresh weight of lettuce plants than DFT system that presented higher plant length.
 Conclusion: Integrated monitoring and control system and IOT provide a wireless sensors network that offered a high capability of accessing huge data anywhere and anytime. Smart management of nutrient solution (TDS, pH, temperature, and level), without smart control, were not useful enough regarding the rapid solution changes and the need for a high response.

Late Ca2+ Sparks and Ripples During the Systolic Ca2+ Transient in Heart Muscle Cells.

The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.

<em>Xenopus</em> Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl+) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H+). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H+ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H+ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.

A new technique for multiple re-use of planar patch clamp chips

The patch clamp technique is widely used for recording the activity of ion channels in single cells and lipid bilayers. Most platforms utilize borosilicate glass configured as a pipette, however more recently planar patch clamp chips have been developed that require less technical expertise. Planar patch clamp chips in systems like the Nanion Port-a-Patch are useful in that they allow more rapid throughput in drug screening studies. This technique also has the ability to perform rapid solution changes from the intracellular side. A current drawback with the planar patch clamp chips is the need to utilize a separate chip for each experiment. This increases the cost of each experiment and is due to the fact that the ∼1μm aperture used for cell attachment is thought to retain cellular debris thereby preventing subsequent cell attachment and formation of GΩ seals. In the present study we have for the first time solved the technical problem of developing a simple protocol for re-use of Nanion planar patch clamp chips. The re-use methodology is demonstrated in whole cell patch clamp studies of HEK-293 cells expressing the electrogenic sodium bicarbonate cotransporter NBCe1-A in protocols involving external and internal solution changes, and CHO-K1 cells with incorporated gramicidin channels.

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na+/Ca2+ exchangers (NCX) constitute a major Ca2+ export system that facilitates the re-establishment of cytosolic Ca2+ levels in many tissues. Ca2+ interactions at its Ca2+ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na+/Ca2+ exchange activity. The structure of the Ca2+-bound form of CBD1, the primary Ca2+ sensor from canine NCX1, but not the Ca2+-free form, has been reported, although the molecular mechanism of Ca2+ regulation remains unclear. Here, we report crystal structures for three distinct Ca2+ binding states of CBD1 from CALX, a Na+/Ca2+ exchanger found in Drosophila sensory neurons. The fully Ca2+-bound CALX-CBD1 structure shows that four Ca2+ atoms bind at identical Ca2+ binding sites as those found in NCX1 and that the partial Ca2+ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca2+ binding at CBD1. The structures also predict that the primary Ca2+ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu455, which coordinates the primary Ca2+ pair, produces dramatic reductions of the regulatory Ca2+ affinity for exchange current, whereas mutagenesis of Glu520, which coordinates the secondary Ca2+ pair, has much smaller effects. Furthermore, our structures indicate that Ca2+ binding only enhances the stability of the Ca2+ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca2+ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Solute Transport in the Transverse Tubules of Cardiac Ventricular Myocytes

Electrical excitation in mammalian cardiac ventricular myocytes underlies the activation of cell-wide Ca2+ release and hence contraction. This process can occur rapidly in relatively large myocytes because transverse tubules (TTs) penetrate deep into the cells. The TT network also permits extracellular solute to be carried into the cell volume and thereby allows for improved inflow and egress of substrates. We have studied TT morphology in relaxed and contracted cells with the aim to characterize transport function and physical properties of the TT system.Using living isolated rat ventricular myocytes, we examined the movement of substrate within the TTs under different conditions using sulforhodamine B and fluorescence imaging. In addition, we examined the TT network with respect to its size, shape and complexity using super-resolution STED (stimulated emission depletion) microscopy. The lipophilic indicator Di-8-ANEPPS was used to identify and characterize the TTs. A rapid bulk solution changer was used to measure the extracellular marker sulforhodamine B concentration changes within the T-tubule matrix at rest and during field stimulation. STED imaging of resting myocytes revealed tubules of about 250 nm in diameter (see related abstract: Wagner et al. 2010). We hypothesized that if TTs collapse and expand dynamically during contraction, solute within the TT network would exchange more rapidly during contractions than when the myocytes are quiescent. Testing this hypothesis by rapid solution change revealed that TT solute exchange was significantly faster during stimulated contraction in single heart cells. Consistent with this finding was the observation that inhibition of contraction by cytochalasin D treatment in paced myocytes reduced the rate of solute exchange. These results suggest that TT solute exchange is accelerated by the mechanical deformation of the TTs during contraction. Future work should enable us to test this hypothesis more directly.

Neurosteroids 3β, 20 (R/S)-pregnandiols decrease offset rate of the GABA-site activation at the recombinant GABA A receptor

Neurosteroids directly modulate ligand gated ion channels such as GABA A receptors. Two such molecules, 3β-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABA A receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5α-pregnan-3β,20(S)-diol (UC1019), 5β-pregnan-3β, 20(R)-diol (UC1020) and PS on the activation onset and offset times of the recombinant GABA A receptor ( rat α 1β 2γ 2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 µM GABA for 10 s, followed by a 80 s washout period. PS (≥ 0.3 µM) moderately increased the slow onset rate ( k on-S) of GABA-response. PS had no significant effects on the fast onset rate ( k on-F). UC1019 and UC1020 decreased the k on-S of the GABA-response in a concentration-dependent manner with no significant effects on the k on-F. Like PS, UC1019 and UC1020 decreased the slow offset rates ( k off-S). In addition, PS increased the fast offset rate ( k off-F) in a concentration-dependent manner, while UC1019 and UC1020 decreased k off-F. The EC 50 of PS to increase k off-F was calculated as 0.47 ± 0.1 µM. The corresponding IC 50 values of UC1019 and UC1020 to decrease k off-F were 5.0 ± 0.5 µM and 8.4 ± 0.9 µM, respectively. These results suggest differential actions of PS and 3β, 20(R/S)-pregnandiols on the offset time course of GABA-site activation.

Xenopus oocyte plasma membrane sheets for FRET analysis

Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca(2+) sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca(2+) additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca(2+) affinity of 1.65 microM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.

Fabrication and Use of High-Speed, Concentric H+- and Ca2+-Selective Microelectrodes Suitable for In Vitro Extracellular Recording

Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H(+) and Ca(2+) are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a "coaxial" or "concentric" inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca(2+)] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca(2+)-selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca(2+)-electrodes were 14.9 +/- 1.3 and 5.3 +/- 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity-dependent, extracellular pH, and [Ca(2+)] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed.

Tools for flash-photolysis experiments on voltage-clamped muscle fibre segments.

An experimental set-up is described that allows the combination of rapid transmembrane voltage changes and photometric calcium recording with the fast photochemical turnover of substances applied externally or intracellularly to cut skeletal muscle fibres. It consists of a double-vaseline-gap system, designed for use with a xenon-flash-lamp device and a dual-wavelength microscope photometer. The pools of the vaseline gap chamber that contain the solutions surrounding the cut ends and the voltage-clamped segment of the muscle fibre are closed and have volumes of 20-50 microl. Thin tubes allow rapid solution change or continuous perfusion in the chamber compartments. Accessory tools were constructed to simplify focussing and measuring the flash-light intensity. A pilot light delivered from a red laser diode is used as a guide beam to target the ultraviolet (UV) flash to the preparation. The light distribution in the focal region and the relative changes in flash intensity with increasing numbers of flashes were quantified with an instrument that integrates the photo-current of a UV-sensitive silicon diode. The function of the set-up was demonstrated by measuring the efficiency of Ca2+ release from DM-nitrophen in quartz capillaries using the Ca(2+)-sensitive dye antipyrylazo III and by recording the flash-induced recovery of L-type calcium currents in muscle fibres blocked by the light-sensitive dihydropyridine drug nifedipine.

Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the gamma-subunit.

1. The gamma-subunit in recombinant gamma-aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA-triggered Cl- currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non-competitive to competitive. To investigate underlying receptor function we studied Zn2- effects on macroscopic and single-channel currents of recombinant alpha 1 beta 2 and alpha 1 beta 2 gamma 2 receptors expressed heterologously in HEK-293 cells using the patch-clamp technique and rapid solution changes. 2. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 +/- 5.6 and 1.41 +/- 0.15 ms, means +/- S.E.M.). Zn2+ (200 microM) reduced the total power almost sixfold and accelerated the slow (23 +/- 2.8 ms, P < 0.05) without altering the fast (1.40 +/- 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 +/- 0.55; Zn2+, 4.9 +/- 0.37, P < 0.05). 5. Zn2+ (500 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 12.2 +/- 2.9%, P < 0.8, n = 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, S = 0.84) relative to that in constant Zn2+. 6. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non-conducting states, site affinity is state-dependent, and the features of state dependence are determined by the gamma-subunit.

Calcium dependence of the apparent rate of force generation in single striated muscle myofibrils activated by rapid solution changes.

Single myofibrils or small groups of myofibrils were isolated from different types of striated muscle: rabbit psoas, frog tibialis anterior, frog atrial and ventricular muscle. The Ca2+ concentration of the solution perfusing the myofibrils was changed within few milliseconds by translating the interface between two flowing streams of solution across the preparations. In all types of myofibrils tested, the time course of force rise in response to maximal activation (pCa 4.75) was approximately monoexponential and nearly superimposable on that observed after a release-restretch protocol applied to the myofibril at the plateau of maximal contractions. This suggests that the kinetics of force development following rapid myofibril activation essentially reflects the kinetics of interaction between contractile proteins. The half time of force rise in response to maximal activation varied among different myofibril types; it was shortest in frog tibialis anterior myofibrils and longest in frog ventricular myofibrils. In all types of myofibril preparations tested the half time of force rise increased with decreasing Ca2+ levels in the activating solution. The finding provides support for a kinetic mechanism of force regulation by Ca2+ in all types of striated muscle. The extent of this Ca2+ effect, however, varied among the different myofibril preparations tested; at 15 degrees C for instance, it was smaller in frog tibialis anterior myofibrils than in the other preparations.

A method for making rapid changes of superfusate whilst maintaining temperature at 37 degrees C.

We have developed a technique for making a rapid solution change, whilst at the same time maintaining the temperature of the preparation at 37 degrees C. It is technically difficult to use rapid solution changes when experiments are performed at normal mammalian body temperature. As a solution is heated from room temperature to 37 degrees C, gas bubbles form in the rapid-flowing solution streams, and these disturb a cell or attached recording pipettes. We describe a system that has been developed to eliminate these problems. We show how to construct the different components of the system, and we have designed an electronic circuit to control solution changes. We have performed tests to characterise the function of this system. Solution flow out of the nozzle of the device (0.88 ml min-1, linear flow velocity 11.6 cm s-1) caused a fall in the steady-state temperature at the experimental preparation of only 0.3 degrees C. The device which takes between 0.5 and 1 s to completely change the superfusate of a single cell, was used routinely with five different experimental solutions. This system may be valuable in studies which require rapid solution changes to be performed at a normal mammalian body temperature.

Amino acid substitutions in the rat Na+, K(+)‐ATPase alpha 2‐subunit alter the cation regulation of pump current expressed in HeLa cells.

1. To study the functional role of negatively charged amino acids (E327 and D925) located in the transmembrane region of the rat alpha 2-isoform of the Na+, K(+)-ATPase (rat alpha 2*) in ion transport, the effects of mutations on external K+ dependence and internal Na+ dependence of pump currents were assessed by the patch-clamp technique in combination with a system for rapid solution changes. 2. Amino acid residues were replaced by glutamine (E327Q) or leucine (D925L) and were introduced into rat alpha 2* cDNA which encodes a ouabain-resistant isoform. These mutant enzymes were stably expressed in HeLa cells. The endogenous ouabain-sensitive HeLa cell Na+, K(+)-ATPase activity was selectively inhibited by 1 microM ouabain present in both the growing media and the assay solution. 3. External K(+)- and internal Na(+)-dependent pump activation was observed in all cells expressing rat alpha 2*, E327Q or D925L; however, the apparent affinities were significantly reduced by the mutations. 4. In E327Q, the activation of pump current was slightly slower than for rat alpha 2*, whereas the deactivation rate was faster. In contrast, D925L produced pump current having dramatically slower activation and deactivation kinetics. 5. These results indicate that these negatively charged amino acids (E327 and D925) are important in cation-induced conformational changes of the protein, which are intermediate steps in the pump mechanism.

Surface potentials measure ion concentrations near lipid bilayers during rapid solution changes

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle.

Effect on the indo‐1 transient of applying Ca2+ channel blocker for a single beat in voltage‐clamped guinea‐pig cardiac myocytes.

1. We used rapid solution changes to investigate the mechanisms which trigger Ca2+ release from the sarcoplasmic reticulum (SR) in guinea-pig ventricular myocytes. We patch-clamped myocytes at 36 degrees C and used indo-1 to monitor intracellular Ca2+. Before each test pulse, we established a standard level of SR Ca2+ load by applying a train of conditioning pulses. 2. We switched rapidly to 32 microM nifedipine (an L-type Ca2+ current (ICa,L) blocker) 8 s before a test pulse, and just after applying nifedipine we applied a ramp depolarization to pre-block Ca2+ channels. We found that ICa,L elicited by the following test pulse was inhibited almost completely (98-99% inhibition). 3. The indo-1 transient elicited by an 800 ms depolarizing pulse showed a rapid initial rise which was inhibited by ryanodine-thapsigargin. This indicated that the rapid rise was due to Ca2+ release from the SR, and therefore provides an index of SR Ca2+ release. 4. In cells dialysed internally with 10 mM Na(+)-containing solution, nifedipine application before a +10 mV test pulse blocked 62% of the rapid initial phase of the indo-1 transient. Calibration curves of indo-1 for intracellular Ca2+ (using a KD of indo-1 for Ca2+ of either 250 or 850 nM, the reported range) indicated that between 67 and 76% of the Ca2+i transient was inhibited by nifedipine. Thus, in cells dialysed with 10 mM Na+ and depolarized to +10 mV, and in the absence of ICa,L, this suggests that another trigger mechanism for SR release is able to trigger between 33 and 24% of the Ca2+i transient. 5. For a given dialysing Na+ concentration, the fraction of indo-1 transient which was inhibited by nifedipine decreased as test potential became more positive. In cells dialysed with 10 mM Na+ and pulsed to +110 mV, 24% of the rapid phase of the indo-1 transient was inhibited by nifedipine (equivalent to between 27 and 37% of the Ca2+i transient). 6. For a given test potential, the fraction of the indo-1 transient which was inhibited by nifedipine decreased as dialysing Na+ concentration increased. In cells dialysed with Na(+)-free solution and pulsed to +10 mV, 84% of the indo-1 transient was inhibited by nifedipine (equivalent to between 88 and 91% of the Ca2+i transient). In contrast, in cells dialysed with 20 mM Na+ and pulsed to +10 mV, 41% of the indo-1 transient was inhibited by nifedipine (equivalent to between 47 and 57% of the Ca2+i transient). 7. Dialysing cells with different Na+ concentrations could lead to a different SR Ca2+ content. We therefore manipulated the conditioning train before each test pulse to change the extent of SR loading. For each dialysing Na+ concentration, we found no change in the degree to which nifedipine blocked the indo-1 transient when SR content was either increased or decreased. 8. The results support the idea that both ICa, L and a second mechanism are able to trigger SR release and the resulting Ca2+i transient. When ICa, L was blocked with nifedipine, the fraction of Ca2+i transient which remained increased with more positive test potential and higher internal Na+. This is consistent with the hypothesis that the second SR trigger mechanism is Ca2+ entry via reverse Na(+)-Ca2+ exchange, elicited by a step change in membrane potential.