The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase.

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS-polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochrondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.     

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

A fluorescence stopped-flow study on troponin labeled with N-ethyl maleimide and N-(p-(2-benzimidazolyl)phenyl) maleimide

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

Cytoplasmic gatekeepers of K+-channel flux: a structural perspective

Recently, rapid progress in our structural knowledge of K(+)-selective channels has started to provide a basis for comprehending the biophysical machinery underlying their electrophysiological properties. These studies have begun to reveal how a diverse array of distinct, cytoplasmically positioned domains affect the activity of associated channels. Some of these establish functional diversity by selectively mediating channel assembly. More importantly, these cytoplasmic domains couple intracellular signals to the gating of their associated pore. New structural insights are providing a clearer understanding of the fundamental molecular mechanisms of these K(+) channels that, in turn, partly underlie complex neurological phenomena.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

Channel-mediated K+ flux in barley aleurone protoplasts

Gibberellic acid (GA₃) stimulates K⁺ efflux from the barley (Hordeum vulgare L. cv. Himalaya) aleurone. We investigated the mechanism of K⁺ flux across the plasma membrane of aleurone protoplasts using patch-clamp techniques. Potassium-ion currents, measured over the entire surface of the protoplast plasma membrane, were induced when the electrochemical gradient for K⁺ was inward (into the cytoplasm). The magnitude and voltage-dependence of this inward current were the same in protoplasts treated with GA₃ and in control protoplasts (no GA₃). Inward currents activated by negative shifts in the membrane potential (E_M) from the Nernst potential for K⁺ (E_K) showed membrane conductance to be a function of the electrochemical gradient (i.e. E_M-E_K). Single-channel influx currents of K⁺ were recorded in small patches of the plasma membrane. These channels had a single-channel conductance of 5–10 pS with 100 mM K⁺ on the inside and 10 mM K⁺ on the outside of the plasma membrane. Single-channel currents, like whole-cell currents, were the same in protoplasts treated with GA₃ and control protoplasts. Voltage-gated efflux currents were found only in protoplasts tha thad been incubated without GA₃. We conclude that K⁺ influx in the aleurone is mediated by channels and these membrane proteins are not greatly effected by GA₃.Abbreviations and symbols F_K Nernst potential for K⁺ - E_M membrane potential - E_rev reversal potential - GA₃ gibberellic acid - K_i concentration of K⁺ inside the cell - K_o concentration of K⁺ outside the cell - R gas constant - S conductance (siemens) - T temperature (^oK) - _i ionic activity coefficient for internal (cytoplasmic) solution - _o ionic activity coefficient for external medium     

Effect of quinine on mitochondrial K+ and Mg++ flux

Quinine decreases rates of unidirectional K+ flux into and out of respiring rat liver mitochondria. K+ efflux is more sensitive to quinine than K+ influx. The data are consistent with the proposal that two separate mechanisms may mediate K+ influx, only one of which is sensitive to quinine. Effects on K+ flux of the stereoisomer quinidine are similar to effects of quinine. The smaller quinuclidine causes at most a slight inhibition of K+ efflux under the same conditions. Mg++ flux exhibits a pattern of inhibition by quinine similar to that of K+ flux. Mg++ efflux is more sensitive to quinine than is Mg++ influx. These and earlier findings indicate marked similarities between liver mitochondrial transport mechanisms for K+ and Mg++.     

The anion transport inhibitor DIDS activates a Ba2+-sensitive K+ flux associated with hepatic exocrine secretion.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor and choleretic organic anion, was used to study the relationship between putative DIDS-sensitive K channels and exocrine secretion in the isolated and bile duct cannulated perfused rat liver. Bile flow, DIDS excretion, and effluent perfusate K+ content were measured. DIDS (125 microM) caused a doubling in bile generation concomitant with its appearance in bile, confirming earlier reports. Furthermore, DIDS induced a transient increase in perfusate K+ concentration that peaked prior to the biliary parameters and, after 10 min, reversed to net uptake that fully compensated for the initial release. The K channel blocker Ba2+ (1 mM) strongly inhibited the release phase along with the accompanying choleresis and DIDS excretion. Ouabain (13.5 microM) alone was choleretic and hyperkalemic and, when applied in combination with DIDS, depressed DIDS excretion, choleresis, and DIDS-sensitive K+ uptake. To obtain further evidence for the presence of DIDS-sensitive K channels K+ flux was measured under the influence of different gradients of the cation. Perfusate K+ at 26 and 80 mM changed the DIDS-activated K+ flux from a transient outward to a sustained inward flux, and both DIDS excretion and bile flow decreased. Mean net K+ flux over 20 min DIDS perfusion changed from -1.3 +/-1.1 micromol/g with 5.9 mM K+ to -1304 +/- 55 micromol/g with 80 mM K+ in the perfusate. K+ efflux was fully and reversibly blocked by Ba2+ and influx was ouabain-insensitive, suggesting that the DIDS-activated K+ flux was channel mediated. The results show that a significant fraction of DIDS-induced bile generation is associated with K+ release that may be mediated by Ba(2+)-sensitive K channels, possibly of the inward rectifying type.