Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. Decreasing extracellular permeant ion concentration decreases outward Na⁺ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na⁺, Li⁺, or hydrazinium) is substituted for a relatively impermeant cation (K⁺, Rb⁺, Cs⁺, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na⁺ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Modulation of Voltage-dependent Properties of a Swelling-activated Cl? Current

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl⁻ current (I _Cl,swell) in BC₃H1 myoblasts. This Cl⁻ current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl⁻ currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg²⁺ concentration or reducing the Cl⁻ concentration of the extracellular solution. Replacing extracellular Cl⁻ by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN⁻ > I⁻ > NO₃ ⁻ > Cl⁻ >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg²⁺, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I _Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.     

Allosteric Gating of a Large Conductance Ca-activated K+ Channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca²⁺. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca²⁺ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca²⁺ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca²⁺ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca²⁺ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

Effects of potassium deficiency on tomato growth and mineral nutrition at the early production stage

The effects of potassium deficiency on hydroponically grown tomato were investigated at the early production stage (23 leaves, 3 trusses). Two types of potassium deficiency were applied : the permanent deficiency lasted for 23 days whereas the 10-day temporary deficiency was followed by a 7-day period of potassium supply resumption.Growth was assessed through non-destructive measurements. Permanent potassium deficiency resulted in growth slow-down before visual symptoms appeared on the adult leaves (leaves 12 to 17), but the older leaves (next to the first truss) were not affected. Temporary potassium deficiency reduced the growth rate, but, after potassium supply resumption, the plants recovered a growth pattern which was similar to that of the control plants. The potassium of the older leaves appeared to be less mobilizable than that present in the adult leaves where the visual deficiency symptoms appeared.Potassium uptake kinetics during the potassium supply resumption period were investigated on the plants submitted to temporary deficiency. In tomato plants which had been temporarily deprived of potassium before being transferred onto a standard nutrient solution, potassium uptake was faster than in the control plants. This result is to be related to the plant ability to recover a normal growth pattern.As a result of the occurrence of K-Mg and K-Na antagonisms, the sum of the cations was maintained at a constant value in some plant organs.     

Isolation and patch clamp measurements of xylem contact cells for the study of their role in the exchange between apoplast and symplast of leaves

Because of their importance for nutrition, a method was developed to patch xylem contact cells in leaves of Vicia faba and maize. Since the lignification of older cells was a major obstacle for isolating protoplasts which could be patched, only young leaves (fourth fully developed leaf) were used. An important step in the isolation of these cells was the infiltration of the leaves and their exposure to enzymes for several hours, allowing mesophyll cells to be removed whilst having most of the xylem contact cells attached to the xylem. Channel activity in cell-attached mode or in excised patches could only be observed if an internal coating of sigmacote was used to block diffusion ions out of the pipette glass. Two different types of K+ channels were identified by measuring the reversal potential at different concentrations of KCl. One channel (SC) had a symmetric IV curve with a high probability of remaining open, irrespective of membrane potential; the other channel was an inward rectifier. The symmetical channel could be blocked weakly by Na+ but it was permeable to .     

Effect of potassium nutrition of rice on rhizosphere redox status

With four soils differing in K supplying power and with four rice cultivars (Oryza sativa L.) differing in K uptake kinetic parameters, the relationship between K fertilizer application and soil redox status in rhizosphere and; the distribution of ferrous iron and other toxic substances on the root surface and in the rhizosphere; and the effect of K supply on uptake of reduced iron by rice plants have been studied.The results show that K application on K-deficient soils reduced the content of active reducing substances and ferrous iron in the soil, raised the soil redox potential in the rhizosphere, increased the Eh value of rice roots and lowered the content of iron in the rice plants. These effects of K varied with different rice cultivars. When no K fertilizer was applied, active reducing substances and ferrous iron in rhizosphere soils were decreased more by the rice cultivars absorbing K strongly (e.g. Shanyou 64) than by cultivars absorbing K weakly (e.g. Zhongguo 91). Therefore, the diminution of the toxic substances by K application in the weakly K-absorbing cultivars was more significant.The observation of a rhizobox separated by a nylon screen showed that appreciably more iron oxides, compared with the control, were deposited at or adjacent to the root surfaces of the rice plant supplied with K fertilizer, fully demonstrating the relationship between K nutrition and the total oxidizing power of rice plants. According to the distribution of active reducing substances and ferrous iron, the oxidizing range of the rice root extended in K application treatment a few centimeters away from the root plane. K application to rice affected the soil redox status in rhizosphere in many ways. The main effect was an increase of the oxidizing power of the rice root. As a result, the value of soil Eh was increased, the contents of active reducing substances and ferrous iron were lowered, as well as the number of oxygen consuming microorganisms.