Page 117 - The Biochemistry of Inorganic Polyphosphates

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Participation in membrane transport 101
to the periplasm. A multi-lane channel is formed between the two polymers, where the outer
wall is lined with solvation oxygens, and the inner wall is girdled by monovalent phosphoryl
anions. At the outer interface, cations are drawn to the ‘mouth’ of the channel by PolyP and
divalent cations are preferentially bound. Ca 2+ occupies most of the binding sites within
the channel and the strong bonds between Ca 2+ and PolyP prevent ion movement, so that
the channel is ‘closed’. The PolyP ‘wire’ of negative charges across the bilayer acts as a
sensor of membrane potential. PolyP reacts to membrane depolarization (or a voltage step
of sufficient strength) by stretching or sliding within the PHB pore, thus dislodging the
resident Ca 2+ and initiating an ion flow. Ca 2+ at the interface then preferentially permeate
into binding cavities at the end of the channel by virtue of their well-suited coordination
geometry and the relatively rapid rate, at which they undergo replacement of hydration
water. Sr 2+ and Ba 2+ are also permeant, but they are not normally found in physiological
systems. These cations have the same coordination geometry as Ca 2+ and, evidently, the
flexible PHB envelope can adjust to accommodate the larger ion size. Since the binding
sites on PolyPs are identical and spaced at frequent intervals, there is no net potential energy
consumption during cation movement within the channel. Segmental motions of the PHB
backbone and librational movements of ester carbonyl oxygens carry Ca 2+ from site to
site in parallel single-file lanes, until the internal concentrations rise to an appropriate level
or the membrane is again polarized. Transition metal cations, particularly trivalent cations
3+
such as La , bind tightly to PolyPs at the interface but have difficulty with entering because
of their unsuitable coordination preferences, and consequently they block the ion flow.
This organization implies that Ca 2+ could be transported out of the cell by extend-
ing the PolyP chain on the cytoplasmic side of the membrane and transporting it through
the PHB pore. As the appended phosphate units move into the PHB channel, Ca 2+ is se-
questered from the cytoplasm, and PolyP–Ca 2+ is exported at the outer face of the membrane
(Figure 7.3).
The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa sub-
units, was found to contain PHB and PolyP (Reusch, 1999b). PHB was detected in both
the tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots
and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay, which
converts PHB into its unique degradation product, crotonic acid. PolyP was detected in
KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain
on SDS-PAGE gels. A band of free PolyP was also visible, suggesting that PolyP is released
when tetramers dissociate. The exopolyphosphatase of S. cerevisiae degraded free PolyP,
but tetramer-associated PolyP was not affected, thus indicating it was inaccessible for the
enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic
assay with polyphosphate kinase. It was suggested that PHB is covalently bound to the
KcsA sub-units, while PolyP is held within the tetramers by ionic forces.
Complexes of PolyP and PHB were found in the membranes of the endoplasmic reticu-
lum and mitochondria of animal cells (Reusch, 1989), which suggests their participation in
the processes of transmembrane transfer. The most intriguing report was that the Ca –
2+
ATPase purified from human erythrocytes contains PolyP and PHB and that the plasma
membrane Ca –ATPase may function as a polyphosphate kinase; it exhibits ATP–PolyP
2+
transferase and PolyP–ADP transferase activities (Reusch et al., 1997). These findings sug-
2+
gest a novel supramolecular structure for the functional Ca –ATPase and a new mechanism
of uphill Ca 2+ extrusion coupled with ATP hydrolysis (Reusch et al., 1997).