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J Physiol (2003), 552.1, pp. 177-183
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.051169
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ABSTRACT |
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Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.
(Received 11 July 2003; accepted after revision 18 August 2003; first published online 22 August 2003)
Corresponding author H. M. Staines: University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. Email: henry.staines{at}physiol.ox.ac.uk
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INTRODUCTION |
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The malaria parasite Plasmodium falciparum induces new permeation pathways (NPP) for a variety of structurally unrelated solutes in the host plasma membrane of infected red blood cells (RBCs) 10-15 h after invasion. The NPP have been characterised previously (using radio-tracer and haemolysis techniques) as anion-selective channels of a single type, which are also permeable to electroneutral and cationic solutes, albeit to a lesser degree (Kirk et al. 1994). They have also been implicated in nutrient uptake, 'waste' removal, volume regulation and ion balance and are generally accepted as excellent chemotherapeutic targets and specific traffic routes for antimalarial agents against the intra-erythrocytic parasite (Kirk, 2001).
The presence of malaria-induced anion channels in the host plasma membrane of mature P. falciparum-infected human RBCs was supported further by the data reported by Desai et al. (2000), using the patch-clamp technique in both the whole-cell and cell-attached patch configurations. They reported a whole-cell conductance in infected cells, which is 150-fold higher than that measured in uninfected cells, rectifies inwardly, has an anion selectivity of I- > Br- > Cl- and a pharmacological profile similar to that reported previously for the NPP (Kutner et al. 1987; Kirk et al. 1993, 1994). At the single channel level, they identified a small conductance (~3 pS) anion-selective channel that is not observed in uninfected RBCs under the same experimental conditions. From these data, Desai et al. concluded that this channel type forms the NPP. Egee et al. (2002), using whole-cell, cell-attached and excised patch configurations, reported broadly similar results, except that they observed a slightly larger conductance (~12 pS) anion channel.
A third study by Huber et al. (2002), using the whole-cell recording configuration, confirmed an inwardly rectifying anion conductance in parasitised RBCs. However, they described also an outwardly rectifying anion conductance and, with additional pharmacological data, concluded that at least two anion-selective channel types must be induced by the parasite (although single channel measurements were not performed). Interestingly, both of the latter two groups performed additional experiments on uninfected cells and reported that several different stimuli (oxidation, phosphorylation and membrane stretch) were able to induce channel activity with properties similar to those observed in infected RBCs, from which they concluded that the intra-erythrocytic parasite activates quiescent, endogenous channels (rather than incorporating parasite-derived channels).
The aim of this study was to investigate possible reasons why discrepancies in whole-cell experimental data have arisen. We have measured whole-cell currents in P. falciparum-infected RBCs, using standard electrophysiological techniques, and show that variations in the preparation of infected RBCs prior to experimentation and the various voltage-clamp protocols used can go some way to clarifying disparities in the reported electrophysiological characteristics.
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METHODS |
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Preparation of cells
Human RBCs (type O), infected with the A4 line of P. falciparum (Roberts et al. 1992), were cultured under 1 % O2-3 % CO2-96 % N2 in RPMI-1640 culture medium, supplemented with (mM): 10 D-glucose, 2 L-glutamine, 40 Hepes, gentamicin sulphate (25 mg l-1), and human serum (8.5 % v/v, pooled from different blood donors). Patch-clamp experiments were carried out using trophozoite-infected RBCs (36-42 h post-invasion), synchronised by a combination of sorbitol haemolysis (Lambros & Vanderberg, 1979) and gelatin flotation (Pasvol et al. 1978). RBCs were drawn directly from the culture (5-20 % parasitaemia) or harvested from the culture by centrifugation on Percoll (yielding suspensions of 80-96 % parasitaemia), as described elsewhere (Kirk et al. 1996). The use of Percoll separation had no obvious effect on whole-cell currents in infected RBCs compared with cells taken directly from culture (data not shown). Prior to experimentation RBCs were washed 3 times in serum-free culture medium followed by 3 washes in bath solution. Single parasitised or uninfected RBCs for patch-clamp experiments were identified by microscopic examination.
Current recordings
Patch pipettes (tip resistances 6-18 M
) were prepared from borosilicate glass capillaries pulled and polished on a Werner Zeitz DMZ programmable puller (Augsburg, Germany). The bath solution contained (mM): 155 NaCl, 1.4 CaCl2, 1 MgCl2, 10 Hepes, 10 glucose, pH 7.4, 320 ± 5 mosmol (kg H2O)-1. The pipette solution was of the same composition, with the one exception that 1.4 mM CaCl2 was replaced with 0.5 mM EGTA.
The ruptured patch whole-cell voltage-clamp configuration was used to record membrane currents (as detailed previously; see for example Sasaki et al. 1999). RBC membrane seals (2-10 G) were obtained by the application of suction to the pipette (1-2 kPa) followed by the imposition of a negative pipette potential (-5 to -30 mV). Cell rupture was attained by a short burst of strong suction and the configuration assessed by a decrease in access resistance and the development of a small capacitance transient. Whole-cell currents were recorded using an Axopatch 200A amplifier, with voltage command protocols generated and the currents analysed using the pCLAMP software suite (Version 8, Axon Instruments Inc., USA). Whole-cell I-V curves were obtained by evoking a series of test potentials (VT) from -100 to +100 mV in 10 mV steps for 300 or 500 ms from a holding potential (VH) of 0 or -30 mV. Data for the construction of I-V curves were measured at 10 ms for each VT for the analysis of 'early' currents and over the last 50 ms of the current records (i.e. 250-300 or 450-500 ms) for the analysis of 'late' currents. All experiments were performed at room temperature. Averaged data are shown as the mean ± S.E.M. In all cases, n denotes the number of cells tested. Significance was assessed using Student's t test.
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RESULTS |
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Effect of holding potential on the whole-cell conductance of malaria-infected RBCs
One clear difference between the studies of Egee et al. (2002) and Huber et al. (2002) is that the former held the cells at 0 mV between test pulses, whereas the latter used predominantly a VH of -30 mV (occasionally -10 mV). Figure 1 illustrates typical whole-cell current recordings, which we have observed in malaria-infected RBCs, and their I-V relationships (analysing late current data) at these two values of VH. Within the first 30 s of obtaining the whole-cell configuration, at either VH, the current recordings were similar (Fig. 1A and B show examples), although, occasionally, a small degree of time-dependent current inactivation was observed at negative VT values (e.g. Fig. 1C). However, as recording continued, there developed in 20 % (3 out of 16) of cells held at 0 mV a striking time-dependent inactivation of inward currents evoked at negative VT values. The change occurred with greater frequency in cells held at -30 mV, where over 60 % (14 out of 23) of cells displayed the effect (representative example shown in Fig. 1C and D). To test if these changes could be reversed, the VH of six cells initially held at -30 mV was changed to 0 mV. Of these six cells, the current change reversed completely in one, while reversing partially in the other five (data not shown).
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Figure 1. Whole-cell current recordings in malaria-infected RBCs A, a representative current recording of an infected RBC directly after attaining the whole-cell configuration, VH = 0 mV. B, as for A except VH = -30 mV. C and D, a representative example of the development of time-dependent current inactivation at negative VT values in an infected RBC, using the whole-cell configuration (in this particular case VH = -30 mV). Note: times shown in the figure represent the times after whole-cell rupture. E, I-V curves for averaged late current data from initial recordings of infected RBCs held at 0 ( | ||
The I-V curves constructed from the averaged data of all initial whole-cell current recordings (n = 51), using late current data, in malaria-infected RBCs held at either 0 or -30 mV showed inward rectification and reversal potentials close to 0 mV. The membrane slope conductances (calculated between -100 and -30 mV) were calculated to be 9.2 ± 0.7 and 10.3 ± 0.8 nS for malaria-infected RBCs held at 0 and -30 mV, respectively (Fig. 1E). No statistically significant difference was found between these conductances (P = 0.4, two-tailed, unpaired Student's t test). However, for those cells where time-dependent current inactivation proceeded to completion (i.e. reaching a steady-state) during the length of the test pulse, the I-V curves for late currents changed from inwardly rectifying to sigmoidal in appearance, whilst retaining their reversal potentials (Fig. 1F).
Effect of serum on the whole-cell conductance of malaria-infected RBCs
The groups also used different protocols for their treatment of the RBCs prior to the electrophysiological investigations. The RBCs used here and also those studied by Egee et al. (2002) were washed at least 3 times in serum-free media. However, Huber et al. (2002) used RBCs taken directly from culture (containing 0.5 % w/v of the lipid-rich bovine serum albumin Albumax II), which were then placed into Petri dishes and perfused (rather than washing). To assess whether these differing preparative protocols modulated significantly the recorded electrophysiological phenotype, we investigated the effects of adding human serum to the bath solution of washed malaria-infected RBCs maintained under whole-cell voltage-clamp.
Figure 2 shows a representative experiment. A malaria-infected RBC (held at 0 mV) showed no alteration in membrane conductance (i.e. time-dependent inward current inactivation was not observed) over the first 6 min of recording (typical inwardly rectifying currents were observed, Fig. 2A and F). At this time, 0.4 % (v/v) human serum was added to the bath solution. This evoked rapid changes in the observed current recordings, which (in this particular cell) were completed after a further 6 min (Fig. 2B). Serum induced an increase in the whole-cell conductance at both positive and negative potentials, with a slight time-dependent inactivation of currents at negative VT values (producing a curvilinear I-V relationship from late current data (Fig. 2F), although steady-state currents at negative VT values were not reached). Changing VH from 0 to -30 mV resulted in the development of a strong time-dependent current inactivation at negative test potentials (including a reduction in the early current amplitudes (i.e. in the first 10 ms) observed at negative VT values), which reached a steady state after ~150 ms. The conductance, when measured from late current data, rectified outwardly and the recording also showed large, decaying tail currents on returning to the VH (Fig. 2C and F). In this case, returning VH to 0 mV completely reversed the effect (Fig. 2D and F) and the further addition of 0.1 mM of the NPP inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; Kirk & Horner, 1995) to the bath solution reduced the conductance by over 90 % (Fig. 2E and F).
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Figure 2. The effect of serum and holding potential on whole-cell current recordings in a malaria-infected RBC A, current recording of an infected RBC immediately before the addition of serum, VH = 0 mV. B, current recording 6 min after addition of human serum (0.4 % v/v). C, current recording 9 min after changing VH from 0 to -30 mV. D, current recording 3 min after changing VH from -30 to 0 mV. E, current recording after addition of 0.1 mM NPPB, VH = 0 mV. F, corresponding I-V curves of late currents under each of the conditions described above (A, | ||
Although addition of serum to the bath modulated both current amplitude and kinetics to some extent in every cell, these effects were variable and were always affected by VH. While some cells in the presence of serum and held at 0 mV showed slight time-dependent inward current inactivation (Fig. 2B), others showed a far higher degree (Fig. 3A). Even so, the latter were still affected by changing VH to -30 mV (Fig. 3B). Other variations included the time required to observe the full effect of changing VH from 0 to -30 mV. Out of 17 cells, this occurred within 60 s for two cells, 2-4 min for 13 cells and between 8 and 9 min for two cells. On returning VH back to 0 mV, the response time was comparable or even faster and the degree of reversibility variable, as complete reversal was observed in only 4 out of 13 cells (compare Fig. 2B with Fig. 2D). It was also noted that the first VT (-100 mV) often accelerated subsequent current inactivation (Fig. 3C).
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Figure 3. The complex nature of whole-cell current recordings in malaria-infected RBCs in the presence of serum A and B, a second example of the effect of serum and holding potential on whole-cell current recordings in an infected RBC. A, current recording after addition of human serum (0.4 % v/v), VH = 0 mV. B, current recording after changing VH from 0 to -30 mV. C, an example of the effect of the initial VT (-100 mV for 500 ms) on a whole-cell current recording in the presence of serum in an infected RBC, VH = 0 mV. D, averaged late outward current (calculated at VT = +100 mV) for cells held at 0 mV ( | ||
From a detailed analysis of all whole-cell experimental data from malaria-infected RBCs in the absence (n = 51) and presence (n = 23) of serum, it is clear that serum increased late outward current amplitudes by approximately 4-fold, independently of the VH (Fig. 3D). At negative test potentials, the effect of serum was shown to nearly double the early inward current amplitudes (i.e. in the first 10 ms) when cells were held at 0 mV, although this effect was not seen at a VH of -30 mV (Fig. 3E). Ultimately, the complex effects observed in currents recorded at negative test potentials made quantitative analysis of the effect of serum on steady-state whole-cell inward currents in malaria-infected RBCs held at 0 mV very difficult. However, it is clear that serum had no effect on late (i.e. steady-state) inward currents, in cells held at -30 mV (Fig. 3F).
In addition to human serum, human plasma (0.4 % v/v), Albumax II (0.025 % w/v) and essentially lipid-free bovine serum albumin (0.025 % w/v) produced similar current changes in malaria-infected RBCs (Fig. 4 shows a representative example of the effect of Albumax II on whole-cell current recordings in malaria-infected RBCs, which can be compared to Fig. 2), while heat-inactivated human serum (10 min at ~100 °C) and serum-free culture medium (used as the bath solution) had no effect (data not shown). Furthermore, all these additions had no effect on the whole-cell currents recorded from uninfected RBCs (data not shown).
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Figure 4. The effect of Albumax II on whole-cell current recordings in a malaria-infected RBC A, current recording of an infected RBC immediately before the addition of Albumax II, VH = 0 mV. B, current recording 6 min after addition of Albumax II (0.025 % w/v). C, current recording after addition of 0.1 mM NPPB. D, corresponding I-V curves of late currents under each of the conditions described above (A, | ||
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DISCUSSION |
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The focus of this study was to clarify some of the possible reasons why groups investigating malaria-infected RBCs with the whole-cell patch-clamp technique have reported contrasting results. In particular, the different experimental conditions used by the groups of Huber et al. and Egee et al. were studied.
Conductance changes in malaria-infected RBC whole-cell patches
Using infected RBCs washed at least three times in serum-free media, initial whole-cell recordings rectified inwardly, as reported by both Desai et al. (2000) and Egee et al. (2002). However, with time, developing inactivation of inward currents was observed in some whole-cell recordings, which was more frequent and more marked on changing to a VH of -30 mV. This sequence of events has not been described previously, neither has the sigmoidal appearance of the steady-state I-V curve for the late current component. It seems likely that this conductance change, once observed, is voltage dependent due to the semi-reversible nature of the change. The fact that full reversal was not observed on every occasion is probably due to the effect of the initial VT of each recording (300 or 500 ms at -100 mV), which on occasion acted in the same way as a -30 mV VH (Fig. 3B shows a similar effect that was recorded in the presence of serum).
The fact that the late current, sigmoidal conductance phenotype develops over time after the whole-cell configuration has been attained suggests that it may be due to the loss of cytosolic components, which are heavily diluted by the pipette solution after cell rupture. However, it is also possible that in attaining the whole-cell configuration there is an instantaneous change to the RBC, which produces the inwardly rectifying recordings (i.e. without time-dependent inward current inactivation) and which takes time to reverse. Therefore, further detailed experiments are required to determine the most physiologically relevant conductance phenotype in these conditions.
Can the effects of VH and serum explain reported differences?
As well as the apparent voltage-dependent changes in membrane conductance discussed above, the addition of human serum (an essential requirement for parasite growth in vitro: Trager & Jenson, 1978) increased both inward and outward whole-cell currents significantly in cells held at 0 mV. There was, however, a complex interplay between the effects of membrane potential and serum, which makes a detailed analysis of the serum effect on inward currents very difficult. It will therefore be important to clarify the mechanisms underlying the changes we have seen. Notwithstanding this, given that the effect of serum was inhibited by NPPB, yet was not observed in uninfected RBCs, it must reflect a modulation of the malaria-induced NPP.
In addition to human serum and human plasma, Albumax II (lipid-rich bovine serum albumin, which also supports parasite growth in vitro (Cranmer et al. 1999) and which is used by the group of Huber et al. (2002)) induced similar effects on whole-cell currents in malaria-infected cells. Therefore the effects of both VH and residual serum components binding to the malaria-infected RBC could explain the outwardly rectifying conductance phenotype reported by Huber et al. (2002).
The physiological importance of these effects
It is clearly possible that the effects of both membrane potential and serum underlie physiological processes that are important to the malaria parasite. Our study shows that quite small changes in holding potential can affect significantly the amplitude and kinetics of the whole-cell conductance of malaria-infected RBCs. The recently reported model for the malaria-infected RBC by Lew et al. (2003) predicts that as the parasite matures the RBC membrane potential drops from approximately -12 mV to -14 mV before increasing again to -2 mV (V. L. Lew, personal communication). With this in mind, variations in the membrane potential of the host plasma membrane in infected RBCs may modulate NPP activity. Furthermore, as stated above, the presence of serum components are a prerequisite for parasite growth. Therefore, one possible role for serum in vivo and in vitro is the proper maintenance of the NPP.
Conclusions
Here we have reported that both changing the holding potential and the addition of serum to the bathing solution can drastically affect the whole-cell conductance of malaria-infected RBCs recorded under voltage clamp. These observations may well help to explain why different conductance phenotypes have been described recently by a number of groups. However, at present the mechanisms underlying these phenotype changes are not understood, neither is their physiological relevance.
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REFERENCES |
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Acknowledgements
This work was supported by the Wellcome Trust (Grant Nos. 066067 and 071662), the French Ministry of Research (PAL+ and PRFMMIP programmes), the French Ministry of Defence (DGA), the WHO/UNDP/World Bank TDR programme, the Foundation Langlois and the Deutsche Forschungsgemeinschaft (La 315/11-1).
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; n = 25) and -30 mV (
; n = 26). F, I-V curves for averaged late current data before (
) and after (
) the development of time-dependent current inactivation (n = 13). Late current data are shown as the mean ± S.E.M. and where not shown error bars lie within the symbols.
; D, 
; E, 
). Note: times shown on the figure represent the times after whole-cell rupture and the I-V curves (shown in F) produced from the data in panels B and D overlie each other.
) in the absence (n = 26) and presence (n = 23) of 0.4 % (v/v) human serum. E, averaged early inward current (calculated at VT = -100 mV) for cells held at 0 mV (