Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.

A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.     

Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion.

The detection of exocytotic fusion in patch-clamped secretory cells depends on measuring an increase in the cell membrane capacitance as new membrane is added to the plasma membrane. However, in the majority of secretory cells, secretory vesicles are too small (< 200 nm in diameter) to cause a detectable signal. We have found that incubations of normal mouse mast cells with the hydrophobic anion dipicrylamine (DPA), increases cell membrane capacitance by about three times. The large capacitive current induced by DPA was voltage-dependent, having a maximum value at -10 mV. The DPA-induced charge movement could be described by a single barrier model in which the DPA molecules move between two stable states in the bulk lipid matrix of the membrane. More importantly, the DPA treatment produced a sevenfold increase in the size of the capacitance steps observed upon the exocytotic fusion of single secretory granules. A similar amplification of DPA on the secretory vesicle capacitance was observed in a cell with larger (< or = 5 microns in diameter) or with smaller secretory granules (< 250 nm in diameter). Additionally, the increased granule membrane capacitance enlarged the transient capacitive discharge measured upon formation of a fusion pore in normal mast cell granules. Our results indicate that hydrophobic ions provide an important tool for high resolution studies of membrane capacitance.     

Patch clamp studies of single intact secretory granules.

The membrane of secretory granules is involved in the molecular events that cause exocytotic fusion. Several of the proteins that have been purified from the membrane of secretory granules form ion channels when they are reconstituted in lipid bilayers and, therefore, have been thought to form part of the molecular structure of the exocytotic fusion pore. We have used the patch clamp technique to study ion conductances in single isolated secretory granules from beige mouse mast cells. We found that the membrane of the intact granule had a conductance of < 50 pS. No abrupt changes in current corresponding to the opening and closing of ion channels were observed, even under conditions where exocytotic fusion occurred. However, mechanical tension or a large voltage pulse caused the breakdown of the granule membrane resulting in the abrupt opening of a pore with an ion conductance of about 1 nS that fluctuated rapidly and could expand to an immeasurably large conductance or close completely. Surprisingly, the behavior of these pores resembled the pattern of conductance changes of exocytotic fusion pores observed in degranulating beige mast cells. This similarity supports the view that the earliest fusion pore is formed upon the breakdown of a bilayer such as that formed during hemifusion.     

Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii

Na+/H+ exchangers (NHEs) are ubiquitous membrane proteins that catalyze the exchange of Na+ for H+ and are critical in pH and cell volume regulation, as well as osmotolerance. In this study, we identify and characterize a novel NHE, TgNHE2, in Toxoplasma gondii. Immunofluorescence studies show that TgNHE2 is localized to the rhoptries, secretory organelles involved in invasion. TgNHE2 is the first intracellular NHE to be characterized in a protozoan parasite and its localization suggests possible roles for the rhoptries in osmotolerance and/or as secretory lysosomes-like granules.     

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.     

Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.     

Rheology of starch–clay nanocomposites

The effects of incorporating various montmorillonite nanoclays into wheat, potato, corn, and waxy corn starch samples were examined by rheology and X-ray diffraction. The nanoclays included the hydrophilic Cloisite Na+ clay as well as the more hydrophobic Cloisite 30B, 10A, and 15A clays. Frequency sweep and creep results for wheat starch–nanoclay samples at room temperature indicated that the Cloisite Na+ samples formed more gel-like materials than the other nanoclay samples. X-ray diffraction results showed no intercalation of Cloisite Na+ clays at room temperature, suggesting that starch granules interacted only with the clay surface and not the interlayer. When the various wheat starch–nanoclay samples were heated to 95 °C, the Cloisite Na+ samples exhibited a large increase in modulus. In contrast, the more hydrophobic nanoclay samples had comparable modulus values to the neat starch sample. These results suggested that during gelatinization, the leached amylose interacted with the Cloisite Na+ interlayer, producing better reinforcement and higher modulus values. X-ray diffraction results supported this interpretation since the data showed greater intercalation of Cloisite Na+ clay in the gelatinized samples. The samples containing wheat and corn starch showed comparable elastic modulus values during gelatinization. However, the potato and waxy corn samples had modulus values that rapidly decreased at higher temperatures.     

Elastic behavior of zymogen granule membranes in response to changes in pH and pCa.

In the process of secretion, the membrane of secretory granules is expected to change its elastic behavior. Elastic modulus of the membrane of zymogen granules, prepared from the rat pancreas acinar cell, was measured by an osmotic swelling method. The elastic modulus of the granule membrane at pCa 8 reduced from the maximal value of 230 dyn/cm at pH 6.0 to almost zero at pH 7.5. In a cytosol of an acinar cell, calcium ions play an important role as a second messenger in secretion. The elastic modulus of the granule membrane reduced in a sigmoidal fashion at pCa between 7.0 and 6.0. This range of pCa corresponds to a physiological rise of free Ca2+ concentrations in the cell cytosol when stimulated by external secretagogues. Reduction of the elastic modulus indicates that the state of the granule membrane switches to a more flexible one in which the granule is easy to appose to the cell plasma membrane and then swell as a final step of exocytosis.     

Methodological approaches to investigation of Na+-Ca2+ exchange in exocrine secretory cells

An analysis of the methodological approaches, that used for investigation of Na+-Ca2+ exchange through the plasma membrane of exciting and secretory cells was presented in this review. Special attention is devoted to identification of Na+-Ca2+ exchange in the model for investigation of Ca2+ transporting systems of secretory cells - salivary glands of Chironomus plumosus L. larvae. With the aim different methods were used: researching of voltage-activated Ca2+-current depending on sodium gradient; studying of changes in the response of secretory glands, incubated in hypo- and hypersodium mediums, and Ca2+ content in their tissues; registration of Na+-Ca2+ exchange current in response to membrane hyper- or depolarisation changes of the membrane potential. And the current dependence on sodium and calcium ion gradient was also studied.     

Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells.

We have monitored L-type Ca2+ channel activity, local cytoplasmic Ca2+ transients, the distribution of insulin-containing secretory granules and exocytosis in individual mouse pancreatic B-cells. Subsequent to the opening of the Ca2+ channels, exocytosis is initiated with a latency < 100 ms. The entry of Ca2+ that precedes exocytosis is unevenly distributed over the cell and is concentrated to the region with the highest density of secretory granules. In this region, the cytoplasmic Ca2+ concentration is 5- to 10-fold higher than in the remainder of the cell reaching concentrations of several micromolar. Single-channel recordings confirm that the L-type Ca2+ channels are clustered in the part of the cell containing the secretory granules. This arrangement, which is obviously reminiscent of the 'active zones' in nerve terminals, can be envisaged as being favourable to the B-cell as it ensures that the Ca2+ transient is maximal and restricted to the part of the cell where it is required to rapidly initiate exocytosis whilst at the same time minimizing the expenditure of metabolic energy to subsequently restore the resting Ca2+ concentration.     

Polyamines and Pectins (I. Ion Exchange and Selectivity)

The ion-binding and -exchange properties of putrescine, spermidine, and spermine on purified walls of carrot (Daucus carota L.) cell suspensions were investigated by producing ion-exchange isotherms and comparing them with the behavior of Na+, Mg2+, and Ca2+. The cation exchange capacity of the carrot cell walls was 0.8 equivalent kg-1 dry matter, and the ionic selectivity sequence of the walls for polyamines followed the sequence spermine4+ > spermidine3+ [almost equal to] Ca2+ > putrescine2+. The polyamines were subjected to only electroselectivity and probably did not induce any favorable supramolecular conformation of pectin like the one induced by Ca2+. Triangular ion exchanges were also performed with three diamines: ethanediamine, butanediamine, and octanediamine. The shorter the diamine, the higher the total adsorption and selectivity of the exchange. The lower selectivity of the cell wall for putrescine was partly attributed to its inability to access and displace Ca2+ from higher affinity sites within dimerized pectic sequences. The polyamine adsorption and exchange on pectic sequences could result in pectic signal modulation in pathogenesis and in differentiation.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Regional Variation in Tissue Composition and Biomechanical Properties of Postmenopausal Ovine and Human Vagina

Objective

There are increasing numbers of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting results. The aim of this study was to compare ovine and human posterior vaginal tissue in terms of histological and biochemical tissue composition and to assess passive biomechanical properties of ovine vagina to further characterise this animal model for pelvic organ prolapse research.

Study Design

Vaginal tissue was collected from ovariectomised sheep (n = 6) and from postmenopausal women (n = 7) from the proximal, middle and distal thirds. Tissue histology was analyzed using Masson''s Trichrome staining; total collagen was quantified by hydroxyproline assays, collagen III/I+III ratios by delayed reduction SDS PAGE, glycosaminoglycans by dimethylmethylene blue assay, and elastic tissue associated proteins (ETAP) by amino acid analysis. Young''s modulus, maximum stress/strain, and permanent strain following cyclic loading were determined in ovine vagina.

Results

Both sheep and human vaginal tissue showed comparable tissue composition. Ovine vaginal tissue showed significantly higher total collagen and glycosaminoglycan values (p<0.05) nearest the cervix. No significant differences were found along the length of the human vagina for collagen, GAG or ETAP content. The proximal region was the stiffest (Young''s modulus, p<0.05), strongest (maximum stress, p<0.05) compared to distal region, and most elastic (permanent strain).

Conclusion

Sheep tissue composition and mechanical properties showed regional differences along the postmenopausal vaginal wall not apparent in human vagina, although the absolute content of proteins were similar. Knowledge of this baseline variation in the composition and mechanical properties of the vaginal wall will assist future studies using sheep as a model for vaginal surgery.     

Shear Elastic Modulus on Patellar Tendon Captured from Supersonic Shear Imaging: Correlation with Tangent Traction Modulus Computed from Material Testing System and Test–Retest Reliability

Characterization of the elastic properties of a tendon could enhance the diagnosis and treatment of tendon injuries. The purpose of this study was to examine the correlation between the shear elastic modulus on the patellar tendon captured from a Supersonic Shear Imaging (SSI) and the tangent traction modulus computed from a Material testing system (MTS) on 8 fresh patellar pig tendons (Experiment I). Test–retest reliability of the shear elastic modulus captured from the SSI was established in Experiment II on 22 patellar tendons of 11 healthy human subjects using the SSI. Spearman Correlation coefficients for the shear elastic modulus and tangent traction modulus ranged from 0.82 to 1.00 (all p<0.05) on the 8 tendons. The intra and inter-operator reliabilities were 0.98 (95% CI: 0.93–0.99) and 0.97 (95% CI: 0.93–0.98) respectively. The results from this study demonstrate that the shear elastic modulus of the patellar tendon measured by the SSI is related to the tangent traction modulus quantified by the MTS. The SSI shows good intra and inter-operator repeatability. Therefore, the present study shows that SSI can be used to assess elastic properties of a tendon.     

Carchesium stalk fibrillar matrix as a highly filled polymer network.

Glycerolated stalks of the sessile peritrich ciliate Carchesium sp. were treated with 10(-6) g ion/1 Ca2+ to disrupt the contractile spasmoneme. The resulting preparation consisted primarily of the fibrillar matrix, a dense extra-cellular meshwork of microfibrils. Some mechanical properties of this preparation have been investigated. The matrix tensile force-extension ratio relation for an initial stretch was characteristic of a soft, swollen polymer network, elastic modulus in young stalks 1.7 X 10(5) Nm-2, in mature stalks 4.0 X 10(5) Nm-2. The higher elastic modulus in mature stalks implies an increase in the interchain cross-link frequency. In young stalks, elastic modulus was found to be independent of the ambient Ca2+ concentration in the threshold range for spasmonemal contraction. Stalk relaxation was pronouncedly irreversible, showing stress softening and permanent hysteresis on repeated loading. Hysteresis was time independent and stiffness was not recovered after four hours at zero strain. Hysteresis was enhanced by repeated loading to the same tensile force. Stress-strain hysteresis at a low extension is characteristic of highly filled polymer networks in which polymer chains are interconnected via rigid filler particles as well as directly cross-linked.     

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle.

Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+.