Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula

The model legume Medicago truncatula possesses a single outward Shaker K⁺ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K⁺ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K⁺ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch‐clamp experiments on root hair protoplasts, besides the Shaker‐type slowly activating outwardly rectifying K⁺ conductance encoded by MtGORK, a second K⁺‐permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock‐out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K⁺ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod‐factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Ionic mechanisms of nonlinearity of the retinal horizontal cell membrane

Changes in ionic conductance lying at the basis of nonlinearity of the current-voltage characteristic curve of the cell (nonsynaptic) membrane of horizontal cells were studied in experiments on the goldfish and turtle retina. All measurements were made during blocking of synaptic transmission by bright light or Co⁺⁺. An increase in the K⁺ concentration led to depolarization and to a reduction of the steepness of the hyperpolarization branch of the current-voltage curve, whereas a decrease in K⁺ had the opposite effect. Changes in the Cl^– or Na⁺ concentrations had no significant effect on membrane potential or on the shape of the current-voltage curve. The principal potential-forming ion in the horizontal cells is thus K⁺; conductance for Cl^– is absent or very low, and conductance for Na⁺ also is evidently small. In the presence of Ba⁺⁺ (2–5 mM) the steepness of the hyperpolarization branch of the current-voltage curve was increased and the whole curve became more linear. It is concluded that nonlinearity of the current-voltage curve of the horizontal cell membrane is due mainly to potential-dependent potassium channels, whose conductance increases during hyperpolarization; this increase in conductance is blocked by Ba⁺⁺. An increase in the Ca⁺⁺ concentration to 20 mM led to an increase in steepness of the depolarization branch of the current-voltage curve, suggesting that depolarization increases membrane conductance for Ca⁺⁺.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 531–539, September–October, 1981.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis

Five-hydroxy tryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl^?ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K⁺ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 m V. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL^?, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K⁺. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5'HT acts on leech body wall muscle cells by increasing the conductance to the Cl^?ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.     

Membrane transport and cytokinin action in root hairs of Medicago sativa

In an effort to develop a cellular model for studying cytokinin action in higher plants, we investigated the effect of cytokinins on growth and membrane transport in root hairs of alfalfa (Medicago sativa␣L.) seedlings. Alfalfa seedlings grown for 24 h in the presence of cytokinins showed increased root hair length and formation of root hairs close to the root cap. Increased growth of root hairs was observed within 10 min of cytokinin application and was accompanied by a hyperpolarization of the plasma membrane. The ion-transport inhibitor diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) blocked both cytokinin-induced root hair growth and hyperpolarization but did not, by itself, alter growth or membrane potential. Finally, hyperpolarization was induced by extracellular cytokinin but not by injection of cytokinin into the cytoplasm. These findings show that root hairs undergo several rapid responses to cytokinin, including changes in membrane transport and growth. We conclude that multiple cytokinin response in root hairs may be mediated by events involving perception and ion transport at the plasma membrane. Received: 18 July 1996 / Accepted: 1 October 1996     

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellateNoctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellateNoctiluca miliaris were examined under voltage-clamp conditions.Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of –80 mV to a potential level more positive than –50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na⁺ concentration was lowered. The transient inward current is assumed to be responsible for the Na⁺-dependent positive spike in non-clamped specimens ofNoctiluca.Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of –20 mV to a potential level more negative than –30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl^– concentration was lowered. The transient outward current is responsible for the Cl^–-dependent negative spike in non-clamped specimens ofNoctiluca.Abbreviations ASW artificial seawater - TRP tentacle regulating potentials - TTX tetrodotoxin     

Effect of Salinity upon Cell Membrane Potential in the Marine Halophyte, Salicornia bigelovii Torr

The electrophysiology of root cells of the marine halophyte, Salicornia bigelovii Torr., has been investigated. Cellular concentrations of K⁺, Cl⁻, and Na⁺ and resulting cell membrane potentials were determined as functions of time and exposure to dilutions of artificial seawater. Treatment of these data by the Nernst criterion suggests that Cl⁻ is actively transported into these root cells, but that active transport need not be invoked to explain the accumulation of Na⁺ at all salinities investigated nor for K⁺ at moderate to high salinities. In low environmental salinity, the cell electropotential of Salicornia root cells was found to respond to inhibitors in a fashion similar to that observed in glycophytes; in high environmental salinity, root cell membrane potential appears to be insensitive to bathing salinity and m-chlorocarbonylcyanide phenylhydrazone induces membrane hyperpolarization, in contrast to the response of glycophytes to such treatments. The fact that measured membrane potentials exceed diffusion potentials for Na⁺, K⁺, and Cl⁻ and the observation of a rapid depolarization by CO in the dark suggests an electrogenic component in Salicornia root cell membrane potentials.     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

The role of ion fluxes in Nod factor signalling in Medicago sativa

Using ion-selective microelectrodes, the basis of Nod factor-induced changes in the plasma membrane potential was analysed by measuring the extracellular free concentrations of Ca²⁺, K⁺, H⁺ and Cl^– in the root hair zone of alfalfa. After addition of the Rhizobium meliloti Nod factor NodRm-IV(C16:2,S) at a concentration of 0.1 μM, a decrease in [Ca²⁺] was observed first, which was followed after a few seconds by an increase of [Cl^–], by an alkalinization, and then by a delayed increase of [K⁺], all of which were transient changes. Simultaneously with the appearance of Cl^– ions in the root hair zone, a decrease in cytosolic [Cl^–] was measured. It was concluded that the depolarization was caused by temporary short-circuiting of the proton pump through the rapid release of Cl^– ions along their steep electrochemical gradient. Since under resting conditions the driving force for K⁺ ions was inwardly directed, their release was delayed until their driving force was inverted. This indicates that K⁺ serves as a charge balance that eventually stops depolarization and initiates repolarization. Since the decrease in [Ca²⁺] was observed seconds before the increase in [Cl^–] and the depolarization, it is argued that Ca²⁺ entering into the cell does not cause the depolarization directly, but might initiate it by triggering the activation of an anion channel that then releases the chloride ions. The observations that the Ca²⁺ ionophore A23187 mimicks the Nod factor response, and that the Ca²⁺ channel antagonist nifedipine inhibits this response, support the idea that Ca²⁺ plays a primary role in the transduction of the Nod signal in alfalfa.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V _e , which attends net Cl^– absorption,J _Cl ^net , and to assess how ADH might augment bothJ _Cl ^met andV _e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R _c /R _bl , and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10^–4 m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V _a . Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10^–4 m luminal furosemide reducedV _e andJ _Cl ^net to near zero values and hyperpolarized bothV _a andV _bl , the voltage across basolateral membranes; however, the depolarization ofV _bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV _a , Thus ADH-dependent increases inV _b were referable to greater depolarizations ofV _bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV _a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV _bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl^– current. Thus an increase in intracellular Cl^– activity may have contributed to the depolarization ofV _bl during net Cl^– absorption, and the intracellular Cl^– activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl^– cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl^– cotransport units in apical plasma membranes.     

Ion channels of intact young root hairs from Medicago sativa

Root hairs are a primary site for nutrient absorption and for initiation of signalling processes linked to variations of the root environment:plant-microbe interactions or abiotic changes. In many of these cases, the earliest detectable response is the modification of plasma membrane transports, detected through alteration of the electrical membrane potential. In spite of this, root hairs have not been extensively used in electrophysiological research so far. Problems with cell shape and current coupling are often prohibitive for microelectrode voltage-clamp on intact root hairs. In the present study, these difficulties have been overcome and the ion channel currents are described for young root hairs from alfalfa seedlings (Medicago sativa cv Sitel). Electrophysiological and pharmacological studies indicated an inward rectifying K⁺ time-dependent current. This current was sensitive to tetraethylammonium and Cs⁺ (10 mM each). Two other currents never shown in root hairs were described: an outward rectifying time-dependent K⁺ current, inhibited by tetraethylammonium and Cs⁺ (10 mM each) allowing K⁺ efflux under strong depolarizations and an instantaneous inward current identified as an anion current, inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and anthracene-9-carboxylic acid (100 μM each). These results should contribute to the understanding of root hair development and of signalling processes in M. sativa root hairs.     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K⁺‐outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K⁺ channels in the membrane. Using gene‐specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus‐induced hyperpolarization‐coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)‐gene and 2 (ac2)‐gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8‐Br‐cAMP and dilution of extracellular [K⁺] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization‐activated inward K⁺ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus‐induced acceleration of ciliary beat in Paramecium.