The Transient Outward Current of Isolated Bovine Adrenal Zona Fasciculata Cells: Comparison between Standard and Perforated Patch Recording Methods

Voltage-clamp experiments were performed on single bovine adrenal fasciculata cells in short-term primary culture using either standard (broken membrane) or perforated whole-cell patch clamp recording. The membrane current measured with the perforated method was dominated by a very stable transient outward current. By contrast, the transient outward current recorded using the standard method was unstable. The reversal potential of the transient outward current varied linearly with the logarithm of [K⁺] _e with a slope of 47 mV per decade. The onset of activation was sigmoidal and was fitted with a power function where n= 4. Time constants ranged from 1 to 4 msec with a maximum at −25 mV. The steady-state activation curve spanned the voltage range −50 to +80 mV without reaching a clear maximum. During a pulse, the current decayed in a biexponential manner. Time constants τ₁ and τ₂ were voltage-dependent and ranged from 50 to 200 msec respectively for a voltage step at +50 mV. The steady-state inactivation was dependent on the conditioning pulse duration. Using short conditioning pulses (1.2 sec), the curve which spanned the voltage range −40 to −20 mV, was 15 mV more positive than that obtained with longer conditioning pulses (60 sec). Time constants of this ``very slow inactivation' process (τ<sub>vs</sub>) determined for voltage steps at −60 and −50 mV were 15 and 10 sec respectively. A ``facilitation process' of the peak current was observed when the duration or the amplitude of conditioning pulses were increased in the voltage range −100 to −50 mV. Recovery from inactivation followed a biexponential time course which seemed a mixture of both inactivation processes. In some experimental conditions, isolated cells were able to produce overshooting action potentials. These results are discussed in relation with the membrane electrogenesis of this cell type. Received: 14 November 1994/Revised: 24 October 1995     

II. Voltage-activated Proton Currents in Human THP-1 Monocytes

Depolarization-activated H⁺-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1 cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H⁺ conductance, g _H, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay, the activation time constant, τ_act was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed mono-exponentially at all potentials. The tail current time constant, τ_tail, was voltage dependent, decreasing with hyperpolarization from 2–3 sec at 0 mV to ∼200 msec at −100 mV. Surprisingly, although τ_act depended strongly on pH _o , τ_tail was completely independent of pH _o . H⁺ currents were inhibited by Zn²⁺. Increasing pH _o or decreasing pH _i shifted the voltage-activation relationship to more negative potentials, tending to activate the g _H at any given voltage. Studied in excised, inside-out membrane patches, H⁺ currents were larger and activated much more rapidly at lower bath pH (i.e., pH _i ). In THP-1 cells differentiated into macrophages, the H⁺ current density was reduced by one-half, and τ_act was slower by about twofold. The properties of H⁺ channels in THP-1 cells and in other macrophage-related cells are compared. Received: 19 September 1995/Revised: 14 March 1996     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Opening of Cardiac Sarcolemmal KATP Channels by Dinitrophenol Separate from Metabolic Inhibition

Opening of ATP-sensitive K⁺ (K_ATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac K_ATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τ_open: 4.8 msec; fast closed time constant, τ_closed(f): 0.33 msec) characteristic of K_ATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open K_ATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of K_ATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced K_ATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996     

Presteady-State Currents of the Rabbit Na+/Glucose Cotransporter (SGLT1)

The rabbit Na⁺/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na⁺, membrane voltage V _m , phlorizin and temperature. Step changes in membrane voltage—from the holding V _h to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state, with time constants τ≈4–20 msec, and were blocked by phlorizin (K _i ≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V _m . The Q-V curves obeyed the Boltzmann relation: the maximal charge Q _max was 4–120 nC; V _0.5, the voltage for 50% Q _max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q _max and z were independent of V _h (between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V _0.5 towards more negative values. Decreasing [Na⁺] _o decreased Q _max, and shifted V _0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na⁺] _o ). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τ_max 8–20 msec. Decreasing [Na⁺] _o decreased τ_max, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τ_max. The maximum temperature coefficient Q ₁₀ for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for rabbit Na⁺/glucose cotransport indicate that charge-movements are due to Na⁺-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na⁺] _o is reduced; (iii) τ_max is decreased with decreasing [Na⁺] _o ; and (iv) the Q-V relations are shifted towards negative voltages as [Na⁺] _o is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model. Received: 12 August 1996/Revised: 30 September 1996     

Large-Conductance Cation Channels in the Envelope of Nuclei from Rat Cerebral Cortex

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K⁺ is 166 pS. The channels are selective for cations with P _K/P _Na= 2. They are neither permeable to, nor gated by Ca²⁺. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles. Received: 12 November 1996/Revised: 18 February 1997     

Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D _eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D _eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P _j ) an estimate of the cytoplasmic diffusion coefficient (D _cyt = 0.7 × 10⁻⁶ cm²/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P _j of 31 × 10⁻⁵ cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P _j of 4.4 × 10⁻⁶ cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. Received: 1 December 1998/Revised: 22 February 1999     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Cl− Channels in Basolateral TAL Membranes XV. Molecular Heterogeneity Between Cortical and Medullary Channels

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl⁻ channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites—threonine 185, threonine 187 and serine 270—which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P _o in basolateral CTAL Cl⁻ channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T_1/2, sec) for ³⁶Cl⁻ efflux in cultured MTAL cells from 82.4 ± 6.8 sec (sem) to 187.8 ± 9.5 sec (n= 5; P= 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T_1/2 for ³⁶Cl⁻ efflux from 80.9 ± 6.3 sec to 191.8 ± 6.5 sec (n= 5; P= 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl⁻ channels mediating net Cl⁻ absorption in mouse MTAL and CTAL, respectively. Received: 16 May 2000/Revised: 30 June 2000     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant α-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, τ^fast _h ) and toxin-bound (slow component, τ^slow _h ) channels. The relative amplitude of τ^slow _h allows an estimate of the IC₅₀ values ∼11 nm. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (τ _h ) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC₅₀= 51 nm). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses. Received: 5 December 1995/Revised: 1 March 1996     

Measurement of Myocardial T1ρ with a Motion Corrected,Parametric Mapping Sequence in Humans

Purpose

To develop a robust T_1ρ magnetic resonance imaging (MRI) sequence for assessment of myocardial disease in humans.

Materials and Methods

We developed a breath-held T_1ρ mapping method using a single-shot, T_1ρ-prepared balanced steady-state free-precession (bSSFP) sequence. The magnetization trajectory was simulated to identify sources of T_1ρ error. To limit motion artifacts, an optical flow-based image registration method was used to align T_1ρ images. The reproducibility and accuracy of these methods was assessed in phantoms and 10 healthy subjects. Results are shown in 1 patient with pre-ventricular contractions (PVCs), 1 patient with chronic myocardial infarction (MI) and 2 patients with hypertrophic cardiomyopathy (HCM).

Results

In phantoms, the mean bias was 1.0 ± 2.7 msec (100 msec phantom) and 0.9 ± 0.9 msec (60 msec phantom) at 60 bpm and 2.2 ± 3.2 msec (100 msec) and 1.4 ± 0.9 msec (60 msec) at 80 bpm. The coefficient of variation (COV) was 2.2 (100 msec) and 1.3 (60 msec) at 60 bpm and 2.6 (100 msec) and 1.4 (60 msec) at 80 bpm. Motion correction improved the alignment of T_1ρ images in subjects, as determined by the increase in Dice Score Coefficient (DSC) from 0.76 to 0.88. T_1ρ reproducibility was high (COV < 0.05, intra-class correlation coefficient (ICC) = 0.85–0.97). Mean myocardial T_1ρ value in healthy subjects was 63.5 ± 4.6 msec. There was good correspondence between late-gadolinium enhanced (LGE) MRI and increased T_1ρ relaxation times in patients.

Conclusion

Single-shot, motion corrected, spin echo, spin lock MRI permits 2D T_1ρ mapping in a breath-hold with good accuracy and precision.     

A Calcium Homeostasis Mechanism Induced by Heterologous Expression of Total RNA from Chicory Leaves in Xenopus Oocytes

Xenopus oocytes were injected with total RNA from chicory leaf tissues and then examined by the voltage-clamp technique. A double-step voltage protocol was used, with an initial hyperpolarization step from the holding potential of −35 to −140 mV followed by a second depolarization step to +60 mV. Two different outward currents were observed, one noninactivating (I _ni ), and one inactivating (I _i ). Only the noninactivating outward current (I _ni ) could be induced by depolarization from −35 to +60 mV. The mean amplitude of I _ni was 2915 ± 848 nA (n= 11). This current, carried by chloride ions, declined nearly to the baseline in 153 ± 64 sec (n= 13), and was highly dependent on intracellular calcium. After the rundown of I _ni , the same oocyte was depolarized from −140 to +60 mV. This protocol induced an inactivating outward current (I _i ) with a mean amplitude of 4461 ± 1605 nA (n= 13). I _i was also carried by chloride ions and dependent on extracellular calcium. I _i was strongly inhibited by 100 μm extracellular La³⁺. These two types of chloride currents were also observed after IP₃ injection in control oocytes. I _ni and I _i were not observed in noninjected oocytes or water-injected oocytes. We suggest that the expression of total chicory leaf tissue RNA in Xenopus oocytes reveals a calcium homeostasis mechanism responsible for calcium mobilization from internal stores and subsequent calcium entry. Received: 22 May 1998/Revised: 2 October 1998     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl− Conductance

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na⁺ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl⁻ into the cell and is negligible when Cl⁻ flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na⁺ current (I _amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I _amil by up to 80%, demonstrating that ENaC is inhibited when Cl⁻ is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I _amil downregulation and the CFTR-mediated plasma membrane Cl⁻ conductance, suggesting a direct correlation. However, I _amil downregulation was not affected when Cl⁻ flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl⁻ reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I _amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca²⁺-sensitive photoprotein aequorin showed that Ca²⁺ is not involved in I _amil downregulation by CFTR, although Ca²⁺ injection into the cytoplasm did inhibit I _amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca²⁺ and is independent of the direction and magnitude of Cl⁻ transport across the plasma membrane. Received: 15 December 1998/Revised: 5 March 1999     

Correlation Between Transepithelial Na+ Transport and Transepithelial Water Movement Across Isolated Frog Skin (Rana esculenta)

In the present work the coupling under short-circuited conditions between the net Na⁺-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (I _sc ) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the I _sc across isolated frog skin is equal to the net transepithelial Na⁺ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the I _sc and TEWM. Under control conditions I _sc was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm⁻² min⁻¹ (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm⁻² min⁻¹. The addition of the Na⁺ channel blocking agent amiloride resulted in a reduction both in I _sc and TEWM, and a linear correlation between I _sc and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na⁺ across the skin. In another series of experiments it was found that there was a linear correlation between I _sc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na⁺ transport and TEWM is caused by local osmosis. Received: 16 October 1996/Revised: 6 March 1997     

细角螺卵子发生的细胞学和超微结构的研究

利用组织切片和透射电镜观察细角螺卵细胞发育的显微和超微结构,结果表明:细角螺卵原细胞期细胞核体积较大,呈椭圆形,核膜明显且有不规则的凹陷,细胞质内出现大量的线粒体和高尔基体.根据卵黄颗粒物的多少和大小可将卵母细胞分为前、中、后三个时期:前期卵母细胞细胞核内染色质浓缩,核仁可见,并出现核周间隙;中期卵母细胞内细胞核移向细胞的一端,核内染色质仍呈高电子密度状态,核仁不明显或消失;后期卵母细胞内的细胞核受挤压形状变得不规则,细胞质内可见少量的线粒体,大量的卵黄颗粒聚集在细胞质中并融合成很大的卵黄球.成熟期卵母细胞卵黄物质多且有较大的脂滴.     

Hypothyroidism Decreases the ATP Sensitivity of KATP Channels from Rat Heart

The effects of thyroid status on the properties of ATP-sensitive potassium channels were investigated. Single-channel recordings were made using excised inside-out membrane patches from enzymatically dissociated ventricular myocytes from hearts of control and thyroidectomized rats and each group was studied with and without administration of thyroid hormone. In patches excised from hypothyroid myocytes the IC₅₀ for ATP inhibition of K_ATP channels was 110 μm. This value was 3-fold higher than the IC₅₀ in control myocytes (43 μm). Treatment of hypothyroid rats to restore physiological levels of thyroid hormone (tri-iodothyronine, T₃), resulted in a return to normal ATP-sensitivity (IC₅₀= 46 μm). In patches from animals rendered hyperthyroid, the IC₅₀ for ATP was 50 μm and this value was not significantly different from the control. There was no difference in the cooperativity of ATP-binding (Hill coefficient, n_H) among control (n_H= 2.2), hypothyroid (n_H= 2.1), T₃-treated (n_H= 2.0) and hyperthyroid groups (n_H= 2.4). The unitary conductance was unchanged and there was no apparent change in intraburst kinetics between examples of single K_ATP channels from control and hypothyroid rats. Action potentials recorded in myocytes from hypothyroid rats were significantly shortened by 50 μm levcromakalim, a K_ATP channel opener (P < 0.001) but unchanged in control myocytes. We conclude that hypothyroidism significantly decreased the ATP-sensitivity of K_ATP channels, whereas the induction of hyperthyroid conditions did not alter the ATP-sensitivity of these channels. Thus, hypothyroidism is likely to have important physiological consequences under circumstances in which K_ATP channels are activated, such as during ischemia. Received: 1 July 1997/Revised: 24 December 1997     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Permeability and Reflection Coefficients of Urea and Small Amides in the Human Red Cell

Measurement of the transport parameters that govern the passage of urea and amides across the red cell membrane leads to important questions about transport of water. It had initially been thought that small protein channels, permeable to water and small solutes, traversed the membrane (see Solomon, 1987). Recently, however, very strong evidence has been presented that the 28 kDa protein, CHIP28, found in the red cell membrane, is the locus of the water channel (see Agre et al., 1993). CHIP28 transports water very rapidly but does not transport small nonelectrolytes such as urea. The irreversible thermodynamic parameter, σ _i , the reflection coefficient, is a measure of the relationship between the permeability of the solute and that of water. If a solute permeates by dissolution in the membrane, σ _i = 1.0; if it permeates by passage through an aqueous channel, σ _i < 1.0. For urea, Goldstein and Solomon (1960) found that σ_urea= 0.62 ± 0.03 which meant that urea crosses the red cell membrane in a water-filled channel. This result and many subsequent observations that showed that σ_urea < 1.0 are at variance with the observation that CHIP28 is impermeable to urea. In view of this problem, we have made a new series of measurements of σ _i for urea and other small solutes by a different method, which obviates many of the criticisms Macey and Karan (1993) have made of our earlier method. The new method (Chen et al., 1988), which relies upon fluorescence of the intracellular dye, fluorescein sulfonate, leads to the corrected value, σ_urea,corr= 0.64 ± 0.03 for ghosts, in good agreement with earlier data for red cells. Thus, the conclusion on irreversible thermodynamic and other grounds that urea and water share a common channel is in disagreement with the view that CHIP28 provides the sole channel for water entrance into the cell. Received: 6 February 1996/Revised: 20 May 1996