CONTROL DE TRÁFICO VEHICULAR

Mitchell defined “proticity” as the flow of protons, in analogy with electricity which is the flow of electrons. Protons and electrons are the smallest particles involved in chemical reactions; the radius of the proton is 1 0"^^ m, whereas the radii of most other ions are

approximately 10'^^ m. The small size of the proton and its large electric field make it exceedingly mobile. “Thus, just as electron-conducting metals are used for the efficient transmission of electric power, so proton-conducting aqueous media are especially suitable

123 for the efficient transmission of protic power” (Mitchell, 1976). Proticity provides the power for accumulation of neurotransmitters in neurosecretory vesicles and in adrenal medullary chromaffin granules.

The internal pH of chromaffin granules was first measured by quenching of fluorescent amines and was estimated to be 5.5 (Bashford et al, 1975a). In the same seminal study which was carried out at Oxford, it was also noted that chromaffin granule membranes possessed ATPase activity whose functional properties were different from those of the mitochondrial ATPase; this ATPase moved protons to the interior of the chromaffin

granules. “Such a movement of protons would lead to the development of both electrical and chemical potential within or across the chromaffin granule membrane; the active uptake of catecholamines may take place by a proton-catecholamine exchange mechanism” (Bashford et al, 1975a). Indeed, inhibition of the ATPase activity was accompanied by inhibition of catecholamine uptake into the granules, confirming the validity of the hypothesis (Bashford et al, 1975b). This novel ATPase was subsequently designated “vacuolar-type ATPase” (V- ATPase; see above).

The chromaffin granule V-ATPase was purified and it was found to be very similar in its properties to the proton-translocating ATPase from mitochondria (Sutton & Apps, 1981). Nonetheless, there were differences which were consistent with Bashford’s suggestion that the chromaffin granules possessed a new form of ATPase which had not been identified hitherto (Sutton & Apps, 1981). The V-ATPase is an extremely complex cellular machine which consists of thirteen different proteins. Complementary cDNAs encoding these proteins were obtained both from mammals and from yeast, in which mutations of the proteins result in phenotypes which can only survive in acidic pH, due to disruption of the V-ATPase activity.

The current model of the three-dimensional structure of V-ATPase is based on the

knowledge which was gained from the structure of mitochondrial F-ATPase (ATP synthase) which was elucidated by John Walker and his colleagues in Cambridge (Abrahams et al,

1994). Using X-ray diffraction at 2.8 A resolution, they found that the catalytic site of the F- ATPase was a globular structure which consisted of three a and three p subunits which were “arranged alternately like the segments of an orange” around a central shaft. Accordingly, the structure of V-ATPase is also believed to consist of multiple subunits which are organised

into four components: a catalytic unit, a “shaft”, a “turbine” and a “hook” (Figure 7.5.3; Nelson & Harvey, 1999). The catalytic unit is believed to consist of alternating A and B subunits which bind and hydrolyse ATP; the A subunit contains a glycine-rich consensus sequence which is a site for binding and hydrolysis of ATP, known as a “Walker motif” (Walker et al, 1982). The B subunit also binds ATP but has no catalytic activity and does not contain a Walker motif. The “turbine” consists of 10-12 copies of a highly lipophihc protein which are arranged in the shape of a barrel which is located in the plasma membrane; this component of the F-ATPase was also crystallised by Walker and his colleagues (Stock et al,

1999). In the F-ATPase, a proton leaving the mitochondrion binds to the carboxyl group in an aspartate residue which is buried in the membrane bilayer, within a subunit of the turbine. As the aspartate becomes protonated, its carboxy terminal helix will rotate. When the

aspartate is deprotonated, the carboxy terminal rotates back to its original position; these alternating movements cause the turbine to rotate (Rastogi & Girvin, 1999). The shaft rotates with the turbine (Sambongi et al, 1999). Rotation of the shaft causes a conformational change in the catalytic unit which remains fixed, enabling the phosphorylation of ADP to ATP. The V-ATPase is believed to function in the reverse direction; hydrolysis of ATP causes conformational changes which rotate the “shaft” which in turn rotates the “turbine” which pumps protons across the membrane. The “hook” consists of two dimeric proteins which are anchored in the plasma membrane and prevent the rotation of the catalytic unit. Thus, the ATP-binding site faces the cytoplasm and the V-ATPase moves protons away from the catalytic unit towards the vesicular lumen which becomes acidic (Nelson & Harvey,

1999). Visual evidence for the rotation of F-ATPase was provided in a remarkable

experiment in which a fluorescent actin filament was attached to the “shaft” as a marker; in the presence of ATP, the filament rotated in an anticlockwise direction when viewed from the membrane side (Noji et al, 1997). Visual evidence for rotation of the turbine was provided in a complementary experiment in which one of the subunits of the turbine was tagged with a fluorescent actin filament; when ATP was added, the turbine rotated in the same

anticlockwise direction and with the same rotational force as had been observed in the case of the shaft (Sambongi et al, 1999). Thus, the rotary torque in the turbine is transmitted by the shaft to the catalytic unit where ATP synthesis or hydrolysis take place (Figure 7.5.3). It is believed that binding of ATP is an energetic step which causes large conformational changes in the molecule; thus, the concentration of ATP determines the rotation rate which in turn determines the rate of catalysis (Yasuda et al, 2 0 0 1).

125 The vesicular membranes of neurosecretory vesicles and adrenal chromaffin granules possess transporter molecules which exchange protons from the acidified interior with neurotransmitters from the cytoplasm, thus functioning as antiporters according to the chemiosmotic hypothesis (Mitchell, 1976). These transporter molecules will be described below (Section 7.5.14).