Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.     

Comparative sequence analysis suggests a conserved gating mechanism for TRP channels

The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate.     

Variable ratio of permeability to gating charge of rBIIA sodium channels and sodium influx in Xenopus oocytes

Whole-cell gating current recording from rat brain IIA sodium channels in Xenopus oocytes was achieved using a high-expression system and a newly developed high-speed two-electrode voltage-clamp. The resulting ionic currents were increased by an order of magnitude. Surprisingly, the measured corresponding gating currents were approximately 5-10 times larger than expected from ionic permeability. This prompted us to minimize uncertainties about clamp asymmetries and to quantify the ratio of sodium permeability to gating charge, which initially would be expected to be constant for a homogeneous channel population. The systematic study, however, showed a 10- to 20-fold variation of this ratio in different experiments, and even in the same cell during an experiment. The ratio of P(Na)/Q was found to correlate with substantial changes observed for the sodium reversal potential. The data suggest that a cytoplasmic sodium load in Xenopus oocytes or the energy consumption required to regulate the increase in cytoplasmic sodium represents a condition where most of the expressed sodium channels keep their pore closed due to yet unknown mechanisms. In contrast, the movements of the voltage sensors remain undisturbed, producing gating current with normal properties.     

Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis

Oocytes of the South African clawed toad Xenopus laevis possess in their plasma membrane a so-called stretch-activated cation channel (SAC) which is activated by gently applying positive or negative pressure (stretch) to the membrane patch containing the channels. We show here that this mechanosensitive channel acted as a spontaneously opening, stretch-independent non-selective cation channel (NSCC) in more than half of the oocytes that we investigated. In 55% of cell-attached patches (total number of patches, 58) on 30 oocytes from several different donors, we found NSCC opening events. These currents were increased by elevating the membrane voltage or raising the temperature. NSCC and SAC currents shared some properties regarding the relative conductances of Na+>Li+>Ca2+, gating behaviour and amiloride sensitivity. Stretch-independent currents could be clearly distinguished from stretch induced SAC currents by their voltage and temperature dependence. Open events of NSCC increased strongly when temperature was raised from 21 to 27 degrees C. NSCC currents could be partly inhibited by high concentrations of extracellular Gd3+ and amiloride (100 and 500 microM, respectively). We further show exemplarily that NSCC can seriously hamper investigations when oocytes are used for the expression of foreign ion channels. In particular, NSCC complicated investigations on cation channels with small conductance as we demonstrate for a 4 pS epithelial Na+ channel (ENaC) from guinea pig distal colon. Our studies on NSCCs suggest the involvement of these channels in oocyte temperature response and ion transport regulation. From our results we suggest that NSCC and SAC currents are carried by one protein operating in different modes.     

Expression of epithelial Na channels in Xenopus oocytes

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride- sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.     

Hidden Markov analysis of mechanosensitive ion channel gating

Patch clamp data from the large conductance mechanosensitive channel (MscL) in E. coli was studied with the aim of developing a strategy for statistical analysis based on hidden Markov models (HMMs) and determining the number of conductance levels of the channel, together with mean current, mean dwell time and equilibrium probability of occupancy for each level. The models incorporated state-dependent white noise and moving average adjustment for filtering, with maximum likelihood parameter estimates obtained using an EM (expectation-maximisation) based iteration. Adjustment for filtering was included as it could be expected that the electronic filter used in recording would have a major effect on obviously brief intermediate conductance level sojourns. Preliminary data analysis revealed that the brevity of intermediate level sojourns caused difficulties in assignment of data points to levels as a result of over-estimation of noise variances. When reasonable constraints were placed on these variances using the better determined noise variances for the closed and fully open levels, idealisation anomalies were eliminated. Nevertheless, simulations suggested that mean sojourn times for the intermediate levels were still considerably over-estimated, and that recording bandwidth was a major limitation; improved results were obtained with higher bandwidth data (10 kHz sampled at 25 kHz). The simplest model consistent with these data had four open conductance levels, intermediate levels being approximately 20%, 51% and 74% of fully open. The mean lifetime at the fully open level was about 1 ms; estimates for the three intermediate levels were 54-92 micros, probably still over-estimates.     

Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes

A three- to five-fold increase in non-cAMP-dependent protein phosphorylation has previously been found to occur in progesterone-treated oocytes shortly before germinal vesicle breakdown (GVBD) or immediately following maturation-promoting factor (MPF) injection. Analysis of phosphoprotein from 32Pi-labeled oocytes by both equilibrium and nonequilibrium two-dimensional gel electrophoresis revealed a large number of qualitative changes in phosphoproteins at GVBD, including both phosphorylation and dephosphorylation events. Time-course studies demonstrated that some of the new phosphoproteins appeared as early as 0.36 GVBD50, and all changes were stable at least through GVBD. The pattern of new phosphoproteins at GVBD was similar in oocytes microinjected with a partially purified preparation of MPF. A number of the new phosphoproteins were heat stable, which may facilitate their purification and characterization. These results support the hypothesis that key regulatory events during oocyte maturation are controlled by protein phosphorylation.     

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.     

A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes.

We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state. The transition is probably one occurring during activation of the channel, as suggested by the fact that one sodium channel mutant demonstrated comparable depolarizing shifts in the voltage dependence of both activation and use-dependent block by TTX. The transition occurred at potentials more negative than those resulting in channel conductance, suggesting that the conformational change that causes use-dependent block by TTX is a closed-state voltage-dependent gating transition.     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Asymmetrical distribution of Ca-activated Cl channels in Xenopus oocytes.

Xenopus oocytes are a popular model system for studying Ca signaling. They endogenously express two kinds of Ca-activated Cl currents, I(Cl-1), and I(Cl-2). I(Cl-1) is activated by Ca released from internal stores and, with appropriate voltage protocols, by Ca influx. In contrast, I(Cl-2) activation is dependent on Ca influx. We are interested in understanding how these two different Cl channels are activated differently by Ca from different sources. One could hypothesize that these channels are activated differently because they are differentially localized near the corresponding Ca source. As an initial investigation of this hypothesis, we examined the distribution of I(Cl-1) and I(Cl-2) channels in the oocyte. We conclude that both I(Cl-1) and (Cl-2) channels are primarily localized to the animal hemisphere of the oocyte, but that capacitative Ca influx occurs over the entire oocyte membrane. Evidence supporting this view includes the following observations: 1) Injection of IP3 into the animal hemisphere produced larger and faster I(Cl-1) responses than injection into the vegetal hemisphere. 2) Exposure of the animal hemisphere to Cl-free solution almost completely abolished I(Cl-1) produced by IP3-induced release of Ca from internal stores or by capacitative Ca entry. 3) Loose macropatch recording showed that both I(Cl-1) and I(Cl-2) currents were approximately four times and approximately three times, respectively, more dense in the animal than in the vegetal hemisphere. 4) Confocal imaging of oocytes loaded with fluorescent Ca-sensitive dyes showed that the time course of activation of I(Cl-1) corresponded to the appearance of the wave of Ca release at the animal pole. 5) Ca release and Ca influx, although twofold higher in the animal pole, were evident over the entire oocyte.     

Mechanism for the selection of nuclear polypeptides in Xenopus oocytes. II. Two-dimensional gel analysis

The role of the nuclear envelope in controlling intracellular protein exchanges was investigated in vivo, by determining the effect of altering nuclear permeability on (a) the protein composition of the nucleoplasm and (b) the nuclear uptake rates of specific endogenous proteins. The nuclear envelopes were disrupted by puncturing oocytes in the region of the germinal vesicle by use of glass needles. Nuclear proteins were analyzed in punctured and control cells by two- dimensional gel electrophoresis, fluorography, and double-labeling techniques. Over 300 nuclear polypeptides were identified in the fluorographs. Of this number, only approximately 10-15 were found to vary between punctured and control nuclei; furthermore, different polypeptides varied in each experiment. These qualitative studies indicate that specific binding within the nucleoplasm, and not selection by the envelope, is the main factor in maintaining the protein composition of the nucleus. The nuclear uptake rates of five individual polypeptides, ranging in molecular weight from 43,000 to 100,000, were analyzed by use of double-labeling procedures. Only one of the polypeptides (actin) entered the nuclei more rapidly after disruption of the envelope. That the nuclear uptake of certain endogenous proteins is unaffected by puncturing demonstrates that passage across the envelope is not a rate-limiting step in the nucleocytoplasmic exchange of these molecules.     

Heteropolymeric potassium channels expressed in Xenopus oocytes from cloned subunits

Voltage-dependent potassium currents were measured in Xenopus oocytes previously injected with RNAs generated in vitro from each of three cloned cDNAs (RBK1, RBK2, and RGK5). The currents differed in their sensitivities to blockade by tetraethylammonium (TEA; respective KDs 0.3, greater than 100, and 10 mM) and in their inactivation during a depolarizing pulse. Injections of RNA combinations (RBK1/RBK2 and RBK1/RGK5) caused currents that had TEA sensitivities different from those expected from the sum, in any proportion, of the two native channels. It is concluded that novel potassium channels are formed by the oocytes injected with two RNAs, presumably by heteropolymerization of subunits; such heteropolymerization would contribute functional diversity to voltage-dependent potassium channels in addition to that provided by a large gene family.     

Biosynthesis of electroplax sodium channels in Electrophorus electrocytes and Xenopus oocytes

We have synthesized the eel electroplax sodium channel core polypeptide in both a cell-free and a frog oocyte system and report it does not possess the unusual electrophoretic properties of the mature, native sodium channel polypeptide isolated from electroplax membranes. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the mature channel polypeptide exhibits both a diffuse banding pattern (microheterogeneity) and an extremely high electrophoretic free mobility. In contrast, the core polypeptide synthesized in vitro or in vivo migrates as a sharp band with a near-normal electrophoretic free mobility (Mr 230,000). The microheterogeneity of the mature peptide has been inferred to result from varying degrees of glycosylation of the channel polypeptide [Miller, J.A., Agnew, W.S., & Levinson, S.R. (1983) Biochemistry 22, 462-470]. We present evidence here that the anomalously high electrophoretic free mobility is due to the binding of large amounts of sodium dodecyl sulfate to posttranslationally modified domains on the protein. In addition, we have followed the posttranslational processing of eel sodium channels in both the eel electrocyte and the frog oocyte. Using lectin binding and Ferguson analysis, we found that the channel was processed relatively rapidly to an intermediate form in the Golgi apparatus that apparently contained fewer carbohydrate and hydrophobic domains than the mature channel. The further addition of carbohydrate and hydrophobic domains, which are required before the channel acquires its characteristic physicochemical properties, proceeded relatively slowly in the electrocyte and appeared not to have occurred to the majority of intermediately processed channels in the frog oocyte.     

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca²⁺ entry through MS channels in Duchenne dystrophy.     

Xenopus connexin38 forms hemi-gap-junctional channels in the nonjunctional plasma membrane of Xenopus oocytes.

A nonselective cation current activated by depolarization (Ic) is present in the nonjunctional membrane of Xenopus oocytes. This current shares a number of properties with hemi-gap-junctional currents induced by exogenous gap-junctional proteins in oocytes and with a nonjunctional current seen in teleost retinal horizontal cells including nonselective permeability to small cations, block by external divalent cations, and slow activation kinetics. Here we study the effects of depleting or overexpressing Cx38 on Ic. Antisense depletion of Cx38 caused a marked reduction in Ic and blocked endogenous gap-junctional coupling in oocyte pairs. Conversely, expression of cloned Cx38 in oocytes increased the amplitude of Ic and enhanced gap-junctional coupling. Furthermore, there appeared to be a close correlation between the temperature sensitivity of Ic and the temperature sensitivity of assembly of endogenous gap-junctional channels in oocyte pairs. These results suggest that Xenopus connexin38 is involved in the generation of Ic.     

Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca²⁺ entry through MS channels in Duchenne dystrophy.