Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.     

Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE

Addition of exogenous Mg²⁺ (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K⁺. Addition of K⁺ (10-50 millimolar) reversed both stromal acidification and K⁺ efflux from the chloroplast caused by Mg²⁺. These data suggested that Mg²⁺ induced reversible H⁺/K⁺ fluxes across the chloroplast envelope. Ca²⁺ and Mn²⁺ (2 millimolar) were as effective as 4 millimolar Mg²⁺ in causing K⁺ efflux from chloroplasts and inhibition of O₂ evolution. In contrast, 10 millimolar Ba²⁺ induced only a small amount of inhibition. The lack of strong inhibition by Ba²⁺ indicated that the effects of divalent cations such as Mg²⁺ cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg²⁺ was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O₂ evolution (43%) induced by Mg²⁺.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+release

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K⁺-permeable channel was coined fast-activating chloroplast cation channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K⁺ conductance domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K⁺ uptake. Nuclear POLLUX ion channels cofunction with Ca²⁺ channels to generate Ca²⁺ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca²⁺ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca²⁺ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca²⁺ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.     

Lidocaine and ATPase inhibitor interaction with the chloroplast envelope

Photosynthetic capacity of isolated intact chloroplasts is known to be sensitive to K⁺ fluxes across the chloroplast envelope. However, little is known about the system of chloroplast envelope proteins that regulate this K⁺ movement. The research described in this report focused on characterizing some of the components of this transport system by examining inhibitor effects on chloroplast metabolism. Digitoxin, an inhibitor of membrane-bound Na⁺/K⁺ ATPases, was found to reduce stromal K⁺ at a range of external K⁺ and inhibit photosynthesis. Scatchard plot analysis revealed a specific protein receptor site with a K_m for digitoxin binding of 13 nanomolar. Studies suggested that the receptor site was on the interior of the envelope. The effect of a class of amine anesthetics that are known to be K⁺ channel blockers on chloroplast metabolism was also studied. Under conditions that facilitate low stromal pH and concomitant photosynthetic inhibition, the anesthetic, lidocaine, was found to stimulate photosynthesis. This stimulation was associated with the maintenance of higher stromal K⁺. Comparison of the effects on photosynthesis of lidocaine analogs which varied in lipophilicity suggested a lipophilic pathway for anesthetic action. The results of experiments with lidocaine and digitoxin were consistent with the hypothesis that a K⁺ channel and a K⁺-pumping envelope ATPase contribute to overall K⁺ flux across the chloroplast envelope. Under appropriate assay conditions, photosynthetic capacity of isolated chloroplasts was shown to be much affected by the activity of these putative envelope proteins.     

Potassium fluxes across the blood brain barrier of the cockroach, Periplaneta americana

Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mM K⁺, an efflux of K⁺ from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN⁻ resulted in an increase in K⁺ efflux across both the ganglia and the connectives. Depletion of K⁺ by chilling the nerve cords in K⁺-free saline was associated with subsequent K⁺ influx across the connectives in K⁺-replete saline at room temperature. There were dramatic increases in K⁺ efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K⁺ fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3 M urea. K⁺ efflux was reduced by the K⁺ channel blockers Ba²⁺ and tetraethylammonium or by exposure to Ca²⁺-free saline and K⁺ efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K⁺ is cycled through a current loop between the ganglia and the connectives and that both the Na⁺/K⁺-ATPase and K⁺ channels are implicated in extracellular K⁺ homeostasis within the central nervous system.     

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.     

K stimulation of ATPase activity associated with the chloroplast inner envelope

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg²⁺ and Mg·ATP complex effects on ATPase activity revealed that any Mg²⁺ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg²⁺ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K⁺. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K⁺. K⁺ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K⁺ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the K_m increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K⁺-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K⁺ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H⁺-ATPase). Such ATPases are thought to be indirectly involved in active K⁺ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K⁺ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo.     

Mitochondrial cation transport: A progress report

This report summarizes recent work in our laboratory aimed at understanding protein-mediated mitochondrial cation transport. We are studying three distinct cation cycles that contain porters catalyzing influx and efflux of cations between cytosol and mitochondrial matrix. Each of these cation cycles plays a major physiological role in the overall energy economy. The K⁺ cycle maintains the integrity of the vesicular structure and includes the K⁺/H⁺ antiporter, the K_ATP channel, and K⁺ leak driven by the high membrane potential. The Ca²⁺ cycle relays the signals calling for modulation of ATP production and includes the Ca²⁺ channel, the Na⁺/Ca²⁺ antiporter, and the Na⁺/H⁺ antiporter. The H⁺ cycle of brown adipose tissue mitochondria provides heat to hibernating and newborn mammals and consists of the uncoupling protein, which catalyzes regulated H⁺ influx.     

Unmasking the mitochondrial K/H exchanger: tetraethylammonium-induced K+-loss.

Mitochondria respiring in media containing 80 mM tetraethylammonium ions lose all of their endogenous K⁺ within 7 minutes. K⁺-loss is associated with uptake of tetraethylammonium ions. K⁺ efflux under these conditions is energy-dependent and electroneutral. It is concluded that tetraethylammonium uptake unmasks the endogenous $\text{K}\text{H}$ exchanger. Considered in relation to the chemiosmotic theory, these results support the existence of a “carrier-brake” mechanism which modulates $\text{K}\text{H}$ exchange to maintain volume homeostasis $\text{in vivo}$.     

Relation between permeability to potassium and sodium ions and fusicoccin-stimulated hydrogen-ion efflux in barley roots

Summary Measurements are described of fusicoccin (FC)-stimulated H⁺ efflux in barley (Hordeum vulgare L.) roots when K⁺ and Na⁺ concentrations were varied. In low-salt roots H⁺ efflux was stimulated in both 5 mM KCl and NaCl. In salt-saturated roots H⁺ efflux was stimulated more effectively in KCl than in NaCl solution. The stimulation of H⁺ efflux thus is parallel with the selectivity of these different root preparations for K⁺ and Na⁺ and with estimates of permeability ratios (P _Na/P _K) determined from electrical measurements. It is suggested that the results support electrogenic coupling between FC-stimulated H⁺ efflux and cation uptake.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Energy-dependent contraction of swollen heart mitochondria--activation by butacaine.

Butacaine and certain other local anesthetics markedly stimulate the rate, extent, and efficiency of respiration-dependent contraction of heart mitochondria in nitrate salts at alkaline pH. The local anesthetics also induce respiratory control associated with contraction (i.e., the elevated rate of respiration during contraction declines to a State 4-like controlled rate when contraction is complete) so that the reaction at alkaline pH closely resembles the rapid and highly efficient process seen at neutral pH. Respiration-dependent contraction appears to be an osmotic response to cation extrusion on an endogenous cation/H⁺ exchanger (G. P. Brierley, M. Jurkowitz, E. Chavez, and D. W. Jung, 1977, J. Biol. Chem.252, 7932–7939). At alkaline pH, net ion extrusion is slow and inefficient due to the elevated permeability of the membrane to monovalent cations through a putative uniport pathway. Butacaine and other local anesthetics seem to decrease influx-efflux cycling of cations at alkaline pH by restricting cation influx through this uniport. Passive swelling at pH 8.3 in nitrate salts indicates that the uniport reaction is sensitive to Ca²⁺ and has a cation-selectivity of Na⁺ > K⁺ > Li⁺. Butacaine does not inhibit passive swelling under these conditions but produces effects identical to those of classical uncouplers and consistent with increased H⁺ conductance and accelerated influx of cations by cation/H⁺ exchange in nonrespiring mitochondria. However, since contraction in respiring mitochondria is inhibited by uncouplers but stimulated by butacaine, it is apparent that butacaine is not an effective proton conductor in energized mitochondria.     

Role of K+ in leaf growth: K+ uptake is required for light-stimulated H+ efflux but not solute accumulation

The stimulation of dicotyledonous leaf growth by light depends on increased H⁺ efflux, to acidify and loosen the cell walls, and is enhanced by K⁺ uptake. The role of K⁺ is generally considered to be osmotic for turgor maintenance. In coleoptiles, auxin‐induced cell elongation and wall acidification depend on K⁺ uptake through tetraethylammonium (TEA)‐sensitive channels (Claussen et al., Planta 201, 227–234, 1997), and auxin stimulates the expression of inward‐rectifying K⁺ channels ( Philippar et al. 1999) . The role of K⁺ in growing, leaf mesophyll cells has been investigated in the present study by measuring the consequences of blocking K⁺ uptake on several growth‐related processes, including solute accumulation, apoplast acidification, and membrane polarization. The results show that light‐stimulated growth and wall acidification of young tobacco leaves is dependent on K⁺ uptake. Light‐stimulated growth is enhanced three‐fold over dark levels with increasing external K⁺, and this effect is blocked by the K⁺ channel blockers, TEA, Ba⁺⁺ and Cs⁺. Incubation in 10 mm TEA reduced light‐stimulated growth and K⁺ uptake by 85%, and completely inhibited light‐stimulated wall acidification and membrane polarization. Although K⁺ uptake is significantly reduced in the presence of TEA, solute accumulation is increased. We suggest that the primary role of K⁺ in light‐stimulated leaf growth is to provide electrical counterbalance to H⁺ efflux, rather than to contribute to solute accumulation and turgor maintenance.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Relationship of Cation Influxes and Effluxes in Yeast

The Na⁺ efflux from Na⁺-rich yeast cells into a cation-free medium is largely balanced by the excretion of organic anions. In the presence of Rb⁺, K⁺, or high levels of H⁺ (pH 3–4), the Na⁺ efflux is increased and the organic anion excretion is suppressed so that stoichiometric cation exchanges occur. H⁺ participates in the exchanges, moving into or out of the cells depending on the external pH and on the concentration of external Rb⁺(K⁺). The total cation efflux is dependent on the external Rb⁺ concentration in a "saturation" relationship, but the individual cations in the efflux stream are not. The discrimination factor in the efflux pathway between H⁺ and Na⁺ is very large (of the order of 10,000), and between Na⁺ and K⁺ considerable (of the order of 50). For the latter pair, the recycling of K⁺ from the cell wall space is an important factor in the discrimination. In addition, the Na⁺ efflux as a function of Na⁺ content follows a sigmoidal curve so that the discrimination factor is increased at high levels of cellular Na⁺. Although the influx and efflux pathways behave as a tightly coupled system, the mechanism of coupling is not entirely clear. A single system with different cation specificities and kinetic behaviors on the inside and outside faces of the membrane could account for the data.     

Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis

Plasma membrane potassium (K⁺) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K⁺ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K⁺ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K⁺ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca²⁺-activated K⁺ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K⁺ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G₁ phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K⁺ channel blocker, did not influence K⁺ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90α, hsp90β, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G₁ phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp.