Opposite Cx32 and Cx26 Voltage-Gating Response to CO2 Reflects Opposite Voltage-Gating Polarity

Previous studies have shown that the V_j-dependent gating behavior of gap junction channels is altered by CO₂ exposure. V_j-dependent channel closure is increased by CO₂ in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V_j (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO₂ affects V_j closure. To test this hypothesis, we have studied the CO₂-mediated changes in V_j gating in channels made of Cx32, Cx26, or a Cx32 mutant (Cx32-N2D) in which asparagine (N) at position 2 was replaced with aspartate (D). With exposure to CO₂, Cx32 channels (negative gaters) show increased V_j-dependent closure, whereas Cx26 channels (positive gaters) respond in the opposite way to V_j. Additionally, Cx32-N2D channels (positive gaters) show decreased V_j closure with exposure to CO₂. The reciprocal Cx26 mutant, Cx26-D2N (negative gater), could not be tested because it did not express functional homotypic channels. The data support the hypothesis that polarity of fast V_j gating determines whether CO₂ increases or decreases the V_j dependent closure of gap junction channels.     

CO<Subscript>2</Subscript> Sensitivity of Voltage Gating and Gating Polarity of GapJunction Channels—Connexin40 and its COOH-Terminus-Truncated Mutant

The CO₂ sensitivity of transjunctional voltage (V _j) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V _j sensitivity, determined by a standard V _j protocol (20 mV V _j steps, 120 mV maximal), decreased significantly with exposure to 30% CO₂. The Boltzmann values of control versus CO₂-treated oocytes were: V ₀ = 36.3 and 48.7 mV, n = 5.4 and 3.7, and G _{j min} = 0.21 and 0.31, respectively. CO₂ also affected the kinetics of V _j-dependent inactivation of junctional current (I _j); the time constants of two-term exponential I _j decay, measured at V _j = 60 mV, increased significantly with CO₂ application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO₂ was also unaffected by CT truncation. There is evidence that CO₂ decreases the V _j sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side of V _j, whereas Cx45 and Cx32 gate at negative V _j, it is likely that V _j behavior with respect to CO₂-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V _j sensor (NH₂-terminus).This revised version was published online in June 2005 with a corrected cover date.     

Is the Voltage Gate of Connexins CO<Subscript>2</Subscript>-sensitive? Cx45 Channels and Inhibition of Calmodulin Expression

The sensitivity of Cx45 channels to CO₂, transjunctional voltage (V _j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage clamp. Cx45 channels are very sensitive to V _j and close with V _j preferentially by the slow gate, likely to be the same as the chemical gate. With a CO₂-induced drop in junctional conductance (G _j), both the speed of V _j-dependent inactivation of junctional current (I _j) and V _j sensitivity increased. With 40-mV V _j-pulses, the of single exponential I _j decay reversibly decreased by 40% during CO₂ application, and G_{j steady state}/G_{j peak} decreased multiphasically, indicating that both kinetics and V _j sensitivity of chemical/slow V _j gating are altered by changes in [H⁺]_i and/or [Ca²⁺]_i. CaM expression was inhibited with oligonucleotides antisense to CaM mRNA. With 15 min CO₂, relative junctional conductance (G _jt/G _jt0) dropped to 0% in controls, but only by 17% in CaM-antisense oocytes. Similarly, V _j sensitivity was significantly lessened in CaM-antisense oocytes. The data indicate that both the speed and sensitivity of V _j-dependent inactivation of the junctional current of Cx45 channels are affected by CO₂ application, and that CaM plays a key role in channel gating.     

Unusual Slow Gating of Gap Junction Channels in Oocytes Expressing Connexin32 or Its COOH-Terminus Truncated Mutant

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V _j)-sensitive gates: fast and slow. Slow V _j gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V _j and is mostly inactive without uncouplers or connexin (Cx) mutations. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of −100 mV V _j pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G _j), measured at each pulse, decreased exponentially by 50−60% (tau = ∼1.2 min). G _jPK dropped more dramatically, such that G _jSS/G _jPK increased from 0.4 to 0.6, indicating a drop in V _j sensitivity. Less striking effects were obtained with –60 mV pulses. During recovery, G _j, measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = ∼7 min). With reversal of V _j polarity, G _jPK briefly increased and G _jSS/G _jPK decreased, suggesting that V _j-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V _j gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V _j behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.     

Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻ channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G _j) and its sensitivity to transjunctional voltage (V _j) by the dual voltage-clamp method. Application of forskolin induced a Cl⁻ current; increased G _j approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V _j gating, monitored by a change in the ratio between G _j steady state and G _j peak (G _jSS/G _jPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G _j and decreased the sensitivity to V _j gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G _j and V _j changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Vasopressin Regulates Water Flow in a Rat Cortical Collecting Duct Cell Line Not Containing Known Aquaporins

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD₁). Transepithelial net water fluxes (J _w ) were recorded every minute in RCCD₁ monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10⁻⁵ m), apical glibenclamide (10⁻⁴ m) and apical BaCl₂ (5 × 10⁻³ m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD₁ cells. AVP stimulates cAMP production and sodium reabsorption in RCCD₁ cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I _sc ) was paralleled by a simultaneous modification of the observed J _w : both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J _w induced by serosal hypertonicity. We can conclude that: (i) transepithelial J _w occurs in RCCD₁ cells in the absence of known renal aquaporins; (ii) the ``water secretory component' observed could be linked to Cl⁻ and K⁺ secretion; (iii) the natriferic response to AVP, preserved in RCCD₁ cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost. Received: 30 December 1999/Revised: 12 October 2000     

Characterization of Whole Cell Chloride Conductances in a Mouse Inner Medullary Collecting Duct Cell Line mIMCD-3

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60 mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN⁻ (2.3), NO⁻ ₃ (1.8), ClO⁻ ₄ (1.7), Br⁻ (1.7), I⁻ (1.6), Cl⁻ (1.0), HCO⁻ ₃ (0.5), gluconate⁻ (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC₅₀ of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN⁻ (2.5), NO⁻ ₃ (1.9), Br⁻ (1.4), I⁻ (1.1), Cl⁻ (1.0), ClO⁻ ₄ (0.5), HCO⁻ ₃ (0.5), gluconate⁻ (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC₅₀ of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca²⁺ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed. Received: 13 June 1995/Revised: 15 September 1995     

Connexin expression systems: To what extent do they reflect the situation in the animal?

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins inXenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP _o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.     

Blocking of Cloned Low-Threshold Ca2+ Channels by Neuroleptic Drugs

We studied the blocking action of neuroleptic drugs, haloperidol, pimozide, and fluspirilene, on three types of cloned low voltage-activated (T-type) Ca²⁺ channels, 1G, 1H, and 1I, functionally expressed in Xenopus oocytes. Fluspirilene and pimozide (members of the diphenylbutylpiperidine group) and haloperidol (belonging to butyrophenones) inhibited Ca²⁺ currents with different values of the K _d constant and maximum intensity of blocking. Effects of the neuroleptics were voltage-dependent and were accompanied by slowing-down of the kinetics of the currents. The mechanism of blocking is probably based on interaction between the neuroleptics and the channels in the activated and inactivated states. The difference in efficiency and specificity of blockade of various T-channel subtypes by neuroleptics should be considered when estimating the therapeutic significance of the tested pharmacological agents.     

ClC-5 Chloride Channel Alters Expression of the Epithelial Sodium Channel (ENaC)

ClC-5 chloride channels and epithelial sodium channels (ENaC) are present in many cell types including airway and retinal epithelia. Since ENaC activity is known to be affected by chloride transport, we co-injected Xenopus oocytes with cRNAs encoding ENaC and ClC-5 to investigate whether channel currents are impacted by heterologous co-expression of these proteins. ClC-5 currents were not detectably affected by co-expression with ENaC, whereas amiloride-sensitive ENaC currents were significantly lower compared to control oocytes expressing ENaC alone. Co-expression of ENaC with cRNA sequences encoding non-conducting fragments of ClC-5 revealed that the amino acid sequence region between positions 347 and 647 was sufficient for inhibition of ENaC currents. Co-expression of ENaC and another transport protein, the sodium dicarboxylate co-transporter (NaDC-1), did not affect ENaC currents. To test whether the inhibitory effects of ClC-5 were specific for ENaC, ClC-5 was also co-expressed with CFTR. CFTR currents were also inhibited by co-expression with ClC-5, whereas ClC-5 currents were unaffected. Western blot analysis of biotinylated oocyte surface membranes revealed that the co-expression of ClC-5 with ENaC, CFTR, or NaDC-1 decreased the abundance of these proteins at the surface membrane. We conclude that overexpression of ClC-5, specifically amino acids 347–647, can alter the normal translation or trafficking of ENaC and other ion transport proteins by a mechanism that is independent of the chloride conductance of ClC-5.     

Cell junctions with funnels in the olfactory mucosa of frogs (Rana temporaria L.)

Summary A characteristic structure in the apical junctional belt of the olfactory epithelium in Rana temporaria is visible in freeze-fracture preparations. This structure is described as a funnel with channel across the junctional belt. It is supposed to represent a possible way for discarding used molecules after stimulation, and to allow the stimulation of free nerve endings in the depth of olfactory epithelia.I wish to thank Prof. C.F. Bardele and Mr. H. Schoppmann for their kind support and technical help     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Role of ligand-gated ion channels in the swimming behaviour of<Emphasis Type="Italic"> Xenopus</Emphasis> tadpoles: experimental data and modelling experiments

The swimming behaviour of lower vertebrates has been used as a model to study the function of simple neuronal circuits. Good examples are the lamprey and the Xenopus tadpole. In these two cases, glutamate-activated NMDA receptors are involved, and the relative importance of the NMDA and non-NMDA receptors as well as the involvement of other ion channels has been studied using a combination of electrophysiological recordings and modelling experiments, but little attention had been paid to their evolution during development. In the present experiments, which have been performed on Xenopus embryos from stages 31 to 42, we have probed the relative importance of the two categories of receptors using selective blockers [respectively dl-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. The sensitivity of the swimming behaviour to APV was found to increase during development and that to CNQX to decrease. Furthermore, it has been observed that the spike activity recorded from the ventral roots is more complex in late embryonic stages that in early embryos. These modifications are associated with changes of the neuronal circuit, some of which correspond to a lengthening of the axon and an increased complexity of the dendritic tree of the motoneurons. We have incorporated these modifications in a simplified model of the central pattern generator built with Neuron software. The results indicate that at least part of the observed changes can be associated with changes in the length of the dendrites and axons.     

Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.