Preprint volume - SIBM

Pre-print Volume - Posters
Topic 2: MARINE ORGANISMS AND ECOSYSTEMS AS MODEL SYSTEMS
A. CHRACHRI 1 , A.R. TAYLOR 1,2 , G.L. WHEELER 1,3 , H. GODDARD 1 , C. BROWNLEE 1
1 The Marine Biological Association, the Laboratory, Citadel Hill, Plymouth PL1 2PB, UK.
aacc@mba.ac.uk
2 University of North Carolina Wilmington, USA.
3 PML, Plymouth, UK.
A VOLTAGE GATED H + -CONDUCTANCE
UNDERLYING PH HOMEOSTASIS
AND CALCIFICATION IN COCCOLITHOPHORIDS
IL VOLTAGGIO “GATED” PER IL FLUSSO DI MEMBRANA
DI IONI H+ COME BASE DELL’OMEOSTASI DEL PH
DELLA CALCIFICAZIONE NEI COCCOLITOFORIDI
Abstract – Intracellular precipitation of calcite in calcifying coccolithophorid such as Coccolithus
pelagicus generate large amounts of H + load in cytosol. Therefore, these cells have a requirement for an
efficient high capacity mechanism for H + excursion. In many animal tissues, it has been demonstrated that
voltage-gated proton (H + ) channels play an important role in cellular defense against acidic stress. In this
report, we have characterized a similar voltage dependent proton conductance in the calcifying
coccolithophorid, Coccolithus pelagicus, using patch-clamp technique. We have also demonstrated that in
voltage-clamp mode, depolarization of these cells from a holding potential of -50 mV to +70 mV caused an
increase in pHi that was accompanied by a sizeable outward conductance. These findings are consistent
with the existence of a conductive H + (equivalent) pathway.
Key-words: coccolithophorid, Coccolithus pelagicus, patch-clamp technique, voltage-gated proton (H + )
channels, pH.
Introduction - Coccolithophorids are photosynthetic marine microalgae that form
extensive blooms in the surface waters of the world’s oceans and are considered to be
the most significant producer of calcite on earth. It has been shown that in Coccolithus
pelagicus calcification occurs in a specialized centrally located intracellular
compartment, the coccolith vesicle, which is derived from the Golgi (Taylor et al.,
2007). The process of intracellular calcification requires efficient and energetically
cost-effective ion transport to the coccolith vesicle as well as intracellular homeostasis
of both Ca 2+ and H + .
Materials and methods – Whole cell patch clamp recordings were conducted at 20 o C.
In some experiments, decalcified cells were either loaded with 300 μM BCECF free
acid in the patch clamp pipette. Changes in intracellular pHi were monitored using a
Zeiss 510 confocal microscope.
Results - Using the patch clamp technique, we characterized a conductance that is
activated by depolarisation more positive than the equilibrium potential for H + . Tail
current analysis revealed that the conductance reversed positively to EK + and ECl - , and
closest to EH + . These data show that the biophysical characteristics of the outward
conductance in C. pelagicus are consistent with those described for animal H + channels
and led us to hypothesize a role in rapid H + efflux during pH homeostasis.
Simultaneous patch clamp and pH imaging demonstrated that in cells that were
clamped at voltages more negative of EH + no change in pHi was observed in response to
sub-threshold depolarization, whereas significant reversible cytoplasmic alkalinisation
41 st S.I.B.M. CONGRESS Rapallo (GE), 7-11 June 2010
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