trans

250 INTRACELLULAR SIGNALING
V-H -ATPase or the V-H -pyrophosphatase.
Moreover, the Na /H exchanger was only
found in acidocalcisomes with V-H -ATPase
activity. In T. cruzi, only a subset of the acidocalcisomes
that can be labeled with antibodies
against the vacuolar PMCA pump also label
with antibodies against the V-H -ATPase.
Different developmental stages also exhibit
biochemical changes in acidocalcisome activities.
The V-H -pyrophosphatase and Na /H
exchanger are each present in procyclic forms
of T. brucei, but are absent from bloodstream
forms. In general, the need for different types of
acidocalcisomes is not clear. Understanding the
role of stored Ca 2 within these organelles and
the physiological mechanisms of Ca 2 release
remain important questions for future research.
Plasma membrane transport systems
Along with the sequestration of Ca 2 into
organelle compartments, the [Ca 2 ] i can be
lowered by transport out of the cell. Ca 2 -
ATPase pumps of the PMCA family are involved
(Figure 11.2). Ca 2 -dependent ATPase activity
has been detected in plasma membrane fractions
from L. donovani, T. brucei, T. cruzi,
Entamoeba invadens and T. gondii. The enzyme
activity from L. donovani and T. cruzi is sensitive
to calmodulin, while the activity from E.
invadens or T. brucei is not. As indicated above,
members of the PMCA family have also been
cloned from T. cruzi, T. gondii, P. falciparum
and E. histolytica. These enzymes generally
localize to multiple cellular compartments.
Ultimately cells rely on extracellular Ca 2 to
initiate signals, or to refill internal pools when
they are emptied. Ca 2 entry across the plasma
membrane is mediated by channels. These
include receptor-operated channels (ROCs),
voltage-operated channels (VOCs), storeoperated
channels (SOCs) and arachidonic
acid-regulated channels (ARCs). In mammalian
cells, VOCs are the best characterized. The 1
subunit from skeletal muscle is capable of
conducting Ca 2 currents, although expression
level and gating properties are altered unless
co-expressed with other channel subunits.
Multiple isoforms of the 1 subunits have
been identified, and these help divide voltagegated
channels into three distinct families.
The 1 subunits each contain four sets of six
transmembrane helices (S1–6). The membraneassociated
loop between helices 5 and 6
contains glutamate residues that provide
selectivity for Ca 2 , while the S4 segment contains
the voltage sensor. Sensitivity to dihydropyridines
is derived from sequences in S6.
Other associated proteins include the 2 complexes,
cytosolic subunit and transmembrane
subunit. Currently, electrophysiological studies
are needed to evaluate channel activity in
parasites. While VOCs have not been described
in protozoan parasites, it is interesting to
note that the flagellum of Chlamydomonas
contains VOC activity. In contrast with VOCs,
the distribution of SOCs is more pervasive.
Nonetheless, the structure is less well understood.
SOC activation is secondary to emptying
of the ER Ca 2 pool, by a process that is
referred to as capacitative Ca 2 entry. A conformational
coupling mechanism that mechanically
connects the ER Ca 2 release channel
with the plasma membrane Ca 2 influx
channel has been proposed. In addition, a
soluble influx factor that is formed on the
surface of the ER and released may also be
involved. In protozoan parasites it is not
known whether cells utilize SOCs and respond
to Ca 2 release from internal pools. The best
characterized Ca 2 influx process is in
T. brucei, and implicates ARCs. In bloodstream
forms of T. brucei, Ca 2 influx across
the plasma membrane can be induced by
exposure to low concentrations of amphiphilic
peptides such as melittin or mastoparan.
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA