John P. Reeves

Mechanisms of active transport in isolated bacterial membrane vesicles: XVIII. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone

Carbonylcyanide m -chlorophenylhydrazone, a well-known uncoupling agent, inhibits lactose and amino acid transport by isolated cytoplasmic membrane vesicles from Escherichia coli and Staphylococcus aureus in the micromolar concentration range and has many of the properties of a typical sulfhydryl reagent. Its inhibitory effects are not alleviated by dilution and washing, but the addition of a number of thiol compounds, dithiothreitol in particular, dramatically blocks and reverses its inhibitory activity. Moreover, incubation of vesicles with this uncoupler diminishes their reactivity towards N -ethylmaleimide, suggesting that the compound blocks sulfhydryl groups in membrane proteins. Neither of these effects are observed with 2,4-dinitrophenol. The results are discussed in terms of chemical and chemiosmotic concepts of energy transduction.

Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger : evidence for the presence of a signal sequence

The Na(+)-Ca2+ exchange carrier was purified from bovine cardiac tissue by a new procedure which relies principally upon anion-exchange chromatography. The purified protein exhibited two major bands on sodium dodecyl sulfate gels, at 120 and 160 kDa. The relative intensities of the two bands could be altered by variations in the procedures used for preparing the samples for electrophoresis, suggesting that they represent two different conformational states of the same protein. The NH2-terminal amino acid sequences of the 120- and 160-kDa bands were identical and agreed closely with a region of the deduced amino acid sequence of the recently cloned canine cardiac exchanger. The NH2-terminal sequence was preceded in the deduced sequence by a 32-residue segment that exhibited the characteristics of a signal sequence; the initial amino acid in the NH2-terminal sequence followed immediately after the predicted cleavage site for the signal sequence. The Na(+)-Ca2+ exchanger appears to be unique among membrane transport carriers in encoding a cleaved signal sequence. The characteristics of the sequences flanking the first putative transmembrane segment of the mature exchanger suggest that the signal sequence is necessary to ensure the correct topological orientation of the exchanger in the membrane.

EVALUATION OF THE CHEMIOSMOTIC INTERPRETATION OF ACTIVE TRANSPORT IN BACTERIAL MEMBRANE VESICLES

In several recent p a ~ e r s , l ~ the chemiosmotic coupling theory has been put forward as a possible mechanism for energy coupling between respiration and transport in bacterial membrane vesicles, This theory has been developed extensively in a number of reviews *, 5-s and will be outlined only briefly here. As visualized in the chemiosmotic model, oxidation of the electron donor is accompanied by the expulsion of protons into the external medium, leading to a pH gradient and/or electrical potential across the membrane. This electrochemical gradient is postulated to be the driving force for inward movement of transport substrates,’ via passive diffusion in the case of lipophilic cations such as the dibenzyldimethylammonium ion,!’, 1 0 via facilitated diffusion in the case of positively charged substrates such as lysine or (in the presence of valinomycin) K+ ions,5 or via coupled movement of H+ with a neutral substrate such as lactose or proline (that is, “symport”) .I1 In instances in which Naf efflux is observed to occur,1* the chemiosmotic model invokes the concept of the Na+-H+ “antiporter,” 3. l.l which is postulated to catalyze electroneutral exchange of internal Na+ with external H+, and vice versa. Furthermore, the inhibitory effects of uncoupling agents such as 2,4-DNP (2,4dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) on the vesicle transport systems are attributed to the ability of these compounds to conduct protons across the membrane and thus collapse the membrane potential.I4 A characteristic feature of the chemiosmotic model is that energy coupling for active transport is visualized as an indirect process mediated by the electrochemical gradient. This hypothesis has a number of important consequences, which are discussed below. Several types of experimental observation have been reported elsewhere by this laboratory that appear to be inconsistent with the chemiosmotic concept as applied to bacterial membrane vesicles.1*9 1.5, Judging from recent reviews by adherents of the theory,’, 17 the significance of these findings with regard to the chemiosmotic model has not been fully appreciated. It is the purpose of this article to review these earlier observations and to report further results

Allosteric Activation of Sodium-Calcium Exchange Activity by Calcium: Persistence at Low Calcium Concentrations

The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation. We assumed that the rate of Ca2+ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca2+ ([Ca2+]i) at which the rate of Ca2+ influx was half-maximal (Kh). After correcting for the effects of mitochondrial Ca2+ uptake and fura-2 buffering, Kh values of ∼300 nM were obtained. After an increase in [Ca2+]i, the activated state of the exchanger persisted following a subsequent reduction in [Ca2+]i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca2+]i, exchange-mediated Ca2+ entry began without a lag period and displayed a linear rate of Ca2+ uptake in most cells; a sigmoidal time course of Ca2+ uptake returned 60–90 s after the transient increase in [Ca2+]i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca2+ pump, suggesting that local Ca2+ gradients contribute to maintaining exchanger activation after the return of global [Ca2+]i to low values.

Ionic regulation of the cardiac sodium-calcium exchanger.

The Na+-Ca2+ exchanger (NCX) links transmembrane movements of Ca2+ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca2+ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca2+ influx under certain conditions. Na+ and Ca2+ ions exert complex regulatory effects on NCX activity. Ca2+ binds to two regulatory sites in the exchangers central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na+ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca2+ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP2) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na+ concentrations also induce an inactivated state of the exchanger (Na+-dependent inactivation) that becomes dominant when cytosolic pH and PIP2 levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca2+ overload due to NCX-mediated Ca2+ influx during ischemia.

Modulation of the Voltage Sensor of L-type Ca2+ Channels by Intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (≥100 μM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channels voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channels central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from ∼0.1 to 100–300 μM sped up the conversion of the gating charge into the negatively distributed mode 10–100-fold. Since the “IQ-AA” mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the “IQ” motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Δ1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+concentration was clamped with gramicidin at ∼20 mM. [Ca2+]iwas varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+regulation between whole cells and membrane patches is unknown.

Solubilization and reconstitution of the sarcolemmal Na+−Ca2+ exchange system of vascular smooth muscle

The Na+-Ca2+ exchange system of the sarcolemma of rat mesenteric artery was solubilized and reconstituted in soybean phospholipid vesicles. In the reconstituted system, the exchange process showed about 4-fold higher specific activity compared to that of native vesicles. The inhibitory effect of monensin and the stimulatory effect of valinomycin in the presence of K+ on Na+ gradient-dependent Ca2+ uptake were preserved and were pronounced in the reconstituted system. The stimulation by valinomycin indicates that the exchange process is electrogenic. Thus, the stoichiometry, the characteristics and the mechanism of action which were difficult to study in the native vesicles can now be determined conveniently using the reconstituted system. Also, solubilization and reconstitution of the exchange system confirms its existence in vascular smooth muscle.

Sodium-calcium exchange in membrane vesicles from Artemia

Membrane vesicles were prepared from Artemia nauplii (San Francisco Bay variety) 45 h after hydration of the dry cysts. Na+-loaded vesicles accumulated up to 10 nmol Ca2+/mg protein when diluted 50-fold into 160 mM KCl containing 15 microM CaCl2. Practically no accumulation of Ca2+ was observed if the vesicles were diluted into 160 mM NaCl instead of KCl, or if they were treated with monensin, a Na+ ionophore, for 30 s prior to addition of CaCl2 to the KCl medium. These observations indicate that the Artemia vesicles exhibit Na-Ca exchange activity. The velocity of Ca2+ accumulation by the vesicles in KCl was stimulated 2.6-fold by the K+ ionophore valinomycin, suggesting that the exchange system is electrogenic, with a stoichiometry greater than 2Na+ per Ca2+. Km,Ca and Vmax values were 15 microM and 7.5 nmol/mg protein.s, respectively. Exchange activity in the Artemia vesicles was inhibited by benzamil (IC50 approximately equal to 100 microM) and by quinacrine (IC50 approximately equal to 250 microM), agents that also inhibit exchange activity in cardiac sarcolemmal vesicles. Unlike cardiac vesicles, however, exchange activity in Artemia was not stimulated by limited proteolysis, redox reagents, or intravesicular Ca2+. This indicates that the two exchange systems are regulated by different mechanisms. Vesicles were prepared from Artemia at various times after hydration of the dry cysts and examined for exchange activity. Activity was first observed at approximately 10 h after hydration and increased to a maximal value by 30-40 h; hatching of the free swimming nauplii occurred at 18-24 h. The results suggest that hatching Artemia nauplii might be a particularly rich source of mRNA coding for the Na+-Ca2+ exchange carrier.

Block of CaV1.2 Channels by Gd3+ Reveals Preopening Transitions in the Selectivity Filter

Using the lanthanide gadolinium (Gd3+) as a Ca2+ replacing probe, we investigated the voltage dependence of pore blockage of CaV1.2 channels. Gd+3 reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd3+ at concentrations used (<1 μM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose–response curves for the two blocking effects of Gd3+ shifted in parallel for Ba2+, Sr2+, and Ca2+ currents through the wild-type channel, and for Ca2+ currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd3+. The correlation indicates that Gd3+ binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range −60 to −45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (−100 to −45 mV) and of use-dependent block (−40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd3+ decreases with concentration of Ba2+, indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd3+ binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.