In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membranebound enzymes. In some of these ATPases, such as Na ϩ ,K ϩ -ATPase and gastric H ϩ ,K ϩ -ATPase, extracellular K ϩ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K ϩ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H ϩ ,K ϩ -ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the α-subunit, have a high SCH 28080-sensitive, but K ϩ -insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K ϩ , but K ϩ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K ϩ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.

Na ؉ ,K ؉ -ATPase and gastric H ؉ ,K ؉ -ATPase are two related enzymes that are responsible for active cation transport. Na ؉ ,K ؉ -ATPase activity is inhibited specifically by ouabain, whereas H ؉ ,K ؉ -ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na ؉ ,K ؉ -ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the ␣-subunit of H ؉ ,K ؉ -ATPase were replaced by their counterparts of the ␣1-subunit of rat Na ؉ ,K ؉ -ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H ؉ ,K ؉ -ATPase were replaced by those of Na ؉ ,K ؉ -ATPase could form a phosphorylated intermediate, but hardly showed a K ؉ -stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na ؉ ,K ؉ -ATPase were also included in this chimeric ATPase, K ؉ -stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34͞56, had obtained a high-affinity ouabain-binding site, whereas the rat Na ؉ ,K ؉ -ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na ؉ ,K ؉ -ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34͞56 chimera, the M1͞M2 loop, however, originates from H ؉ ,K ؉ -ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1͞M2 loop of chimera HN34͞56 results in a decreased ouabain affinity. This indicates that although the M1͞M2 loop affects the ouabain affinity, binding occurs when the M3͞M4 and M5͞M6 hairpins of Na ؉ ,K ؉ -ATPase are present.

To investigate the role of Glu820, located in transmembrane domain M6 of the alpha-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820-->Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40-100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10-20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy) imidazo [1,2a]-pyridine (SCH28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the alpha-subunit.

Several mutations of residues Glu795 and Glu 820 present in M5 and M6 of the catalytic subunit of gastric H ؉ ,K ؉ -ATPase have resulted in a K ؉ -independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E 1 conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu 343 , present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K ؉ -activated ATPase activity and an E 2 preference and a Gln giving K ؉ -independent ATPase activity and an E 1 preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E 2 7 E 1 equilibrium was still dominated by the 820 residue: a Glu giving E 2 and a Gln giving E 1 . This indicates that the preference for the E 1 conformation of the E820Q mutation is independent of an active dephosphorylation process.

Gene therapy is a rapidly developing field in which recombinant nucleic acid sequences are introduced to individuals to regulate, repair, replace, add or delete a genetic sequence. Recombinant adeno-associated viral (AAV) vectors, especially AAV2, are frequently used in gene therapy. Knowledge on the biodistribution and potential shedding of AAV2 is crucial to evaluate the risks of infection with the viral vector for the patient and the environment. Literature was analysed for biodistribution and shedding data for AAV2. Preclinical and clinical studies were included with a focus on the influence of the administration route on spreading. Based on biodistribution and shedding data, a qualitative model for the biodistribution and shedding of AAV2 related to the administration route is presented. It is concluded that biodistribution and shedding of AAV2 depends on the route of administration. Some routes lead to local biodistribution and thus to no shedding or shedding via one route only. Other routes lead to systemic biodistribution and to shedding via several excretion routes. The qualitative model presented can help to determine the possible biodistribution in the body and the risk of shedding via the different excretion routes. In addition, it can help to predict the different shedding routes after a certain administration route of AAV2 and thus in deciding which studies are warranted or which safety precautions are needed after administration to patients.

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Recently a molecular model was proposed lor the binding site of the antagonist 3M -)-/V-(2,3-dihydro-l-mediyl-2-oxo-5~phenyl-l H-1,4-benzodia/epine-3 s I) I // mdole 2-c^'hoxamide (deva/epide) on the cholecystokinin-A (CCK A) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129 14X). Fifteen amino acids were identified, including hydrophilic ones such as Seru '\ Asivw and S e r'71\ that might interact with the caihoxamide moiety in deva/epide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin <2(> 33)^peptkle amide (CCK^S) mid CCK-H-evoked (V 1 mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity. £9 1997 Elsevier Science B.V.

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.