The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or guanosine 5'-[beta gamma-imido]triphosphate to the cells, but not adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pHsensitive residues. 2. Extracellular Cd¥ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1·0 ± 0·1 mÒ and a Hill coefficient of 2·0 ± 0·3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6·8 ± 0·1 and a Hill coefficient for proton binding of 3·0 ± 0·3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6·4 ± 0·1 and a Hill coefficient for proton binding of 1·0 ± 0·2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd¥ (IC50, 3·0 ± 0·3 mÒ) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd¥ binding site was reduced to 1·4 ± 0·3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd¥, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does alter Cd¥ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.

1. The administration of glucagon to fed rats by intraperitoneal injection, or the perfusion of livers from fed rats with glucagon by the method of Mortimore [Mortimore (1963) Am.J. Physiol. 204, 699--704] was associated with increases of 15- and 5-fold respectively, in the time for which a given load of exogenous Ca2+ is retained by mitochondria subsequently isolated from the liver. This effect of glucagon was (a) also induced by N6O2'-dibutyryl cyclic AMP, (b) completely blocked by cycloheximide, (c) relatively slow in onset (15--60 min) and (d) associated with a stimulation of about 20% in the rates of ADP-stimulated oxygen utilization and Ca2+ transport measured in the presence of succinate. 2. Perfusion of livers with glucagon resulted in the isolation of mitochandria which showed a 50% increase, no significant change and a 40% increase in the concentrations of endogenous Ca, Mg and Pi respectively, when compared with mitochondria isolated from control perfused livers. 3. The administration of insulin or adrenaline to fed rats induced increases of 10- and 8-fold respectively, in the time for which Ca2+ is retained by isolated liver mitochondria. Perfusion of livers with insulin had no effect on mitochondrial Ca2+ retention time. 4. The perfusion of livers from starved rats with glucagon, or the administration of either glucagon or insulin to starved rats, increased by about 2.5- and 15-fold respectively, the time for which isolated mitochondria retain Ca2+. 5. Mechanisms which may be responsible for the observed alterations in Ca2+-retention time are discussed.

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (⌬D13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and ⌬D13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys 877 to Arg 894 .In mammalian skeletal muscle, the voltage-gated chloride channel, ClC-1, is responsible for the greater proportion of the resting conductance, which acts to stabilize the membrane potential against unwanted perturbations. Its structure, as predicted by hydropathy analysis (1) and in common with other members of the large ClC family, includes 13 relatively hydrophobic domains (termed D1 to D13), most of which probably span the membrane. There is good evidence, however, that D4 and D13 are located entirely extracellularly and intracellularly, respectively (2, 3), with the implication that close to half the amino acids of human ClC-1 (hClC-1) 1 must form an extensive cytoplasmic tail (Gln 574 -Leu 988 ). The D13 domain, which is conserved among all eukaryotic ClC channels (4), is situated in this tail, about two-thirds of the way to the carboxyl terminus (Leu 840 -Ile 870 ). Mutations in ClC-1 have been associated with both dominant and recessive forms of myotonia (e.g. Refs. 5 and 6) characterized by abnormal, sustained firing of action potentials that result in prolonged involuntary muscle contractions. In one (R894X) of two myotonic mutations identified in the carboxyl tail (7, 8) the final 95 amino acids of the protein (8) are lost. Functional analysis of this mutant, expressed in Xenopus oocytes, showed a large reduction in chloride currents, which could account for the myotonic symptoms (9). In similar studies (4), chloride currents were totally lost from human ClC-1 truncated at Ser 720 (G721X) and at Gln 597 ...