Interactions between selenium and sulphur nutrition in Arabidopsis thaliana

Selenium (Se) is an essential plant micronutrient, but is toxic at high tissue concentrations. It is chemically similar to sulphur (S), an essential plant macronutrient. The interactions between Se and S nutrition were investigated in the model plant Arabidopsis thaliana (L.) Heynh. Arabidopsis plants were grown on agar containing a complete mineral complement and various concentrations of selenate and sulphate. The Se/S concentration ratio in the shoot ([Se](shoot)/[S](shoot)) showed a complex dependence on the ratio of selenate to sulphate concentration in the agar ([Se](agar)/[S](agar)). Increasing [S](agar) increased shoot fresh weight (FW) and [S](shoot), but decreased [Se](shoot). Increasing [Se](agar) increased both [Se](shoot) and [S](shoot), but reduced shoot FW. The reduction in shoot FW in the presence of Se was linearly related to the shoot Se/S concentration ratio. These data suggest (i) that Se and S enter Arabidopsis through multiple transport pathways with contrasting sulphate/selenate selectivities, whose activities vary between plants of contrasting nutritional status, (ii) that rhizosphere sulphate inhibits selenate uptake, (iii) that rhizosphere selenate promotes sulphate uptake, possibly by preventing the reduction in the abundance and/or activity of sulphate transporters by sulphate and/or its metabolites, and (iv) that Se toxicity occurs because Se and S compete for a biochemical process, such as assimilation into amino acids of essential proteins.     

The Arabidopsis‐related halophyte Thellungiella halophila: boron tolerance via boron complexation with metabolites?

Tolerance to boron (B) is still not completely understood. We tested here the hypothesis that Thellungiella halophila, an Arabidopsis thaliana-related 'extremophile' plant, with abundance of B in its natural environment, is tolerant to B, and examined the potential mechanisms of this tolerance. With 1-10 mm B applied ([B](ext)) to Thellungiella and Arabidopsis grown in hydroponics, the steady-state accumulated B concentration ([B](int)) in the root was below [B](ext), and was similar in both, suggesting both extrude B actively. Whether grown in soil or hydroponically, the shoot [B](int) was higher in Arabidopsis than in Thellungiella, suggesting more effective net B exclusion by Thellungiella root. Arabidopsis exhibited toxicity symptoms including reduced shoot fresh weight (FW), but Thellungiella was not affected, even at similar levels of shoot-accumulated [B](int) (about 10 to 40 mm B in 'shoot water'), suggesting additional B tolerance mechanism in Thellungiella shoot. At [B](ext) = 5 mm, the summed shoot concentration of the potentially B-binding polyhydroxyl metabolites (malic acid, fructose, glucose, sucrose and citric acid) in Arabidopsis was below [B](int) , but in Thellungiella it was over twofold higher than [B](int) , and therefore likely to allow appreciable 1:2 boron-metabolite complexation in the shoot. This, we suggest, is an important component of Thellungiella B tolerance mechanism.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Arabidopsis Alcohol Dehydrogenase Expression in Both Shoots and Roots Is Conditioned by Root Growth Environment

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.     

Influx and accumulation of Cs(+) by the akt1 mutant of Arabidopsis thaliana (L.) Heynh. lacking a dominant K(+) transport system.

An extensive literature reports that Cs(+), an environmental contaminant, enters plant cells through K(+) transport systems. Several recently identified plant K(+) transport systems are permeable to Cs(+). Permeation models indicate that most Cs(+) uptake into plant roots under typical soil ionic conditions will be mediated by voltage-insensitive cation (VIC) channels in the plasma membrane and not by the inward rectifying K(+) (KIR) channels implicated in plant K nutrition. Cation fluxes through KIR channels are blocked by Cs(+). This paper tests directly the hypothesis that the dominant KIR channel in plant roots (AKT1) does not contribute significantly to Cs(+) uptake by comparing Cs(+) uptake into wild-type and the akt1 knockout mutant of Arabidopsis thaliana (L.) Heynh. Wild-type and akt1 plants were grown to comparable size and K(+) content on agar containing 10 mM K(+). Both Cs(+) influx to roots of intact plants and Cs(+) accumulation in roots and shoots were identical in wild-type and akt1 plants. These data indicate that AKT1 is unlikely to contribute significantly to Cs(+) uptake by wild-type Arabidopsis from 'single-salt' solutions. The influx of Cs(+) to roots of intact wild-type and akt1 plants was inhibited by 1 mM Ba(2+), Ca(2+) and La(3+), but not by 10 microM Br-cAMP. This pharmacology resembles that of VIC channels and is consistent with the hypothesis that VIC channels mediate most Cs(+) influx under 'single-salt' conditions.     

Is the ABA concentration in the sap collected by pressurizing leaves relevant for analysing drought effects on stomata? Evidence from ABA-fed leaves of transgenic plants with modified capacities to synthesize ABA.

Most studies on the role of ABA in the stomatal response of the whole plant to drought rely on a good estimate of ABA concentration in xylem sap. In this report, varying volumes of sap (V(sap)) were collected by pressurizing leaves cut from several lines of N. plumbaginifolia with modified capacities to synthesize ABA. Leaves were fed with solutions of known ABA concentration ([ABA](solution) from 0-500 micromol m(-3)) for 2-3 h before sap collection. ABA concentration in extruded sap ([ABA](sap)) was compared with [ABA](solution). In low-volume extracts (less than 0.35 mm(3) cm(-2) leaf area) collected from leaves of well-watered plants, [ABA](sap) was close to [ABA](solution). For all lines, [ABA](sap) decreased with increasing V(sap). The same dilution effect was observed for leaves pressurized just after sampling on droughted plants, suggesting, as for detached leaves fed with ABA, that [ABA](sap) in low-volume extracts approximated well with the concentration of ABA entering leaves still attached on droughted plants. However, ABA-fed leaves sampled from droughted plants yielded higher [ABA](sap) than ABA-fed leaves sampled from well-watered plants. [ABA](sap) was also increased, although very slightly, when leaves were preincubated in highly enriched ABA solution. This indicates that some leaf ABA contributed to the ABA concentration returned in the extruded sap. Consistently, [ABA](sap) in medium-volume extracts (0.35-0.65 mm(3) cm(-2) leaf area) was lower for leaves sampled on under-producing lines than on the wild type. Despite these distortions between [ABA](solution) and [ABA](sap) in medium-volume extracts, stomatal conductance of ABA-fed leaves closely correlated with [ABA](sap) with a similar relationship in all cases, whilst relationships with [ABA](solution) were more scattered.     

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.     

Characterization of the mechanisms of action and nitric oxide species involved in the relaxation induced by the ruthenium complex

Nitric oxide (NO) plays an important role in the control of vascular tone. NO donors have therapeutic use and the most used NO donors, nitroglycerin and sodium nitroprusside have problems in their use. Thus, new NO donors have been synthesized to minimize these undesirable effects. Nytrosil ruthenium complexes have been studied as a new class of NO donors. trans-[RuCl([15]aneN(4))NO](2+), induces vasorelaxation only in presence of reducing agent. In this study, we characterized the mechanisms of vasorelaxation of trans-[RuCl([15]aneN(4))NO](2+) in denuded rat aorta and identified which NO forms are involved in this relaxation. We also evaluated the effect of this NO donor in decreasing the cytosolic Ca(2+) concentration ([Ca(2+)]c) of the vascular smooth muscle cells. Vasorelaxation to trans-[RuCl([15]aneN(4))NO](2+) (E(max): 101.8 +/- 2.3%, pEC(50): 5.03 +/- 0.15) was almost abolished in the presence of the NO* scavenger hydroxocobalamin (E(max): 4.0 +/- 0.4%; P < 0.001) and it was partially inhibited by the NO(-) scavenger L-cysteine (E(max): 79.9 +/- 6.9%, pEC(50): 4.41 +/- 0.06; P < 0.05). The guanylyl cyclase inhibitor ODQ reduced the E(max) (57.7 +/- 4.0%, P < 0.001) and pEC(50) (4.21 +/- 0.42, P < 0.01) and the combination of ODQ and TEA abolished the response to trans-[RuCl([15]aneN(4))NO](2+). The blockade of voltage-dependent (K(v)), ATP-sensitive (K(ATP)), and Ca(2+)-activated (K(Ca) K(+) channels reduced the vasorelaxation induced by trans-[RuCl([15]aneN(4))NO](2+). This compound significantly reduced [Ca(2+)]c (from 100% to 85.9 +/- 3.5%, n = 4). In conclusion, our data demonstrate that this NO donor induces vascular relaxation involving NO* and NO(-) species, that is associated to a decrease in [Ca(2+)]c. The mechanisms of vasorelaxation involve guanylyl cyclase activation, cGMP production and K(+) channels activation.     

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

Natural variation of Arabidopsis response to nitrogen availability

Our understanding of plant growth in response to nitrogen (N) supply is mainly based on studies of mutants and transformants. This study explored the natural variability of Arabidopsis thaliana first to find out its global response to N availability and secondly to characterize the plasticity for growth and N metabolism among 23 genetically distant accessions under normal (N+), limited (N-), and starved (N0) N supplies. Plant growth was estimated by eight morphological traits characterizing shoot and root growth and 10 metabolic parameters that represented N and carbon metabolism. Most of the studied traits showed a large variation linked to genotype and nutrition. Furthermore, Arabidopsis growth was coordinated by master traits such as the shoot to root ratio of nitrate content in N+, root fresh matter and root amino acids in N-, and shoot fresh matter together with root thickness in N0. The 23 accessions could be gathered into four different groups, according to their growth in N+, N-, and N0. Phenotypic profiling characterized four different adaptative responses to N- and N0. Class 1 tolerated N limitation with the smallest decrease in shoot and root biomass compared with N+, while class 2 presented the highest resistance to N starvation by preferential increased root growth, huge starch accumulation, and high shoot nitrate content. In contrast, class 3 plants could tolerate neither N limitation nor N starvation. Small plants of class 4 were different, with shoot biomass barely affected in N- and root biomass unaffected in N0.     

Alkali grass resists salt stress through high [K+] and an endodermis barrier to Na+

In order to understand the salt-tolerance mechanism of alkali grass (Puccinellia tenuiflora) compared with wheat (Triticum aestivum L.), [K(+)] and [Na(+)] in roots and shoots in response to salt treatments were examined with ion element analysis and X-ray microanalysis. Both the rapid K(+) and Na(+) influx in response to different NaCl and KCl treatments, and the accumulation of K(+) and Na(+) as the plants acclimated to long-term stress were studied in culture- solution experiments. A higher K(+) uptake under normal and saline conditions was evident in alkali grass compared with that in wheat, and electrophysiological analyses indicated that the different uptake probably resulted from the higher K(+)/Na(+) selectivity of the plasma membrane. When external [K(+)] was high, K(+) uptake and transport from roots to shoots were inhibited by exogenous Cs(+), while TEA (tetraethylammonium) only inhibited K(+) transport from the root to the shoot. K(+) uptake was not influenced by Cs(+) when plants were K(+) starved. It was shown by X-ray microanalysis that high [K(+)] and low [Na(+)] existed in the endodermal cells of alkali grass roots, suggesting this to be the tissue where Cs(+) inhibition occurs. These results suggest that the K(+)/Na(+) selectivity of potassium channels and the existence of an apoplastic barrier, the Casparian bands of the endodermis, lead to the lateral gradient of K(+) and Na(+) across root tissue, resulting not only in high levels of [K(+)] in the shoot but also a large [Na(+)] gradient between the root and the shoot.     

Interpretation of concentration-dependence in aggregation kinetics

Aggregation processes are analyzed by two kinetic models, the random polymerization model and the nucleation-dependent polymerization model. A kinetic equation for the random polymerization model can be derived analytically, revealing the relation between the initial monomer concentration ([M]0), the rate constant (k(a)), time (t), the yield of detectable aggregate ([F]), and the critical aggregation number (m). However, time-course curves for the nucleation-dependent polymerization model can be obtained by numerical calculation. It is found that lag time (t(d)) and half-time (t1/2) are proportional to [M](-1) in the random polymerization model, while t(d) and t1/2 are proportional to [M1](-s) (1 < s < n; n is nucleus size) at the lower concentration and are less dependent on [M1] at the higher concentration in the nucleation-dependent polymerization model.     

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

Origins of the transient anterior-posterior asymmetry in the frog lens fiber potential

The origin of the transient asymmetry of intracellular resting potentials between the anterior and posterior lens fibers was investigated in the isolated American bullfrog lens by a conventional microelectrode technique. In high K+, Rb+, Cs+, or NH+4 test solution applied only to the lens anterior or posterior side, anterior fibers depolarized at a slower rate than posterior ones. After a long exposure, however, the transient potential difference disappeared. The magnitude of the depolarizations of the lens fibers was in the order of K+ greater than Rb+ greater than Cs+ greater than NH+4. The resting potentials plotted as a function of external K+ concentrations ([K]0) were in agreement with Nernst equation predictions with a slope of 58 mV/decade ion concentration change. A small Na+ permeability is unmasked at a [K]0 less than 10 mM. It was concluded that the transient difference measured in potentials of anterior and posterior lens fibers on increasing external K+, Rb+, Cs+ or NH+4 depends on the anterior epithelial cell layer, which is a diffusional barrier for ions penetrating into the lens interior.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Na/K pump current and [Na](i) in rabbit ventricular myocytes: local [Na](i) depletion and Na buffering

Na/K pump current (I(pump)) and intracellular Na concentration ([Na](i)) were measured simultaneously in voltage-clamped rabbit ventricular myocytes, under conditions where [Na](i) is controlled mainly by membrane transport. Upon abrupt pump reactivation (after 10-12 min blockade), I(pump) decays in two phases. Initially, I(pump) declines with little [Na](i) change, whereas the second phase is accompanied by [Na](i) decline. Initial I(pump) sag was still present at external [K] = 15 mM, but prevented by [Na](i) approximately 100 mM. Initial I(pump) sag might be explained by subsarcolemmal [Na](i) ([Na](SL)) depletion produced by rapid Na extrusion and I(pump). Brief episodes of pump blockade allowed [Na](SL) repletion, since peak postblockade I(pump) exceeded I(pump) at the end of previous activation (without appreciably altered global [Na](i)). The apparent K(m) for [Na](i) was higher for continuous I(pump) activation than peak I(pump) (14.1 +/- 0.2 vs. 11.2 +/- 0.2 mM), whereas that based on d[Na](i)/dt matched peak I(pump) (11.6 +/- 0.3 mM). [Na](SL) depletion (vs. [Na](i)) could be as high as 3 mM for [Na](i) approximately 18-20 mM. A simple diffusion model indicates that such [Na](SL) depletion requires a Na diffusion coefficient 10(3)- to 10(4)-fold below that expected in bulk cytoplasm (although this could be subsarcolemmal only). I(pump) integrals and [Na](i) decline were used to estimate intracellular Na buffering, which is slight (1.39 +/- 0.09).     

The pacemaker current in cardiac Purkinje myocytes

It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time- dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization- activated inward current i(f).