(Received for publication, September 26, 1995; and in revised form, December 6, 1995)
From the
The mechanism of spermidine release from Xenopus oocytes was examined by measuring release of radioactive
[H]spermidine under different ionic conditions,
and under voltage-clamp. In normal solution (2 mM K
), the efflux rate is less than 1% per hour, and
is stimulated
2-fold by inclusion of Ca
(1
mM) in the incubation medium. Spermidine efflux is stimulated
10-fold in high [K
] (KD98) solution. In
KD98 solution, efflux is strongly inhibited by divalent cations (K
for Ba
block of
spermidine efflux is
0.1 mM), but not by
tetraethylammonium ions or verapamil. Spermidine efflux rates were not
different between control oocytes and those expressing HRK1 inward
rectifier K
(Kir) channels. When the membrane
potential was clamped, either by changing external
[K
] in oocytes expressing HRK1, or by
2-microelectrode voltage-clamp, spermidine efflux was shown to be
strongly dependent on voltage, as expected for a simple
electrodiffusive process, where spermidine
is the
effluxing species. This result argues against spermidine diffusing out
as an uncharged species, or in exchange for similarly charged
counterions. These results are the first conclusive demonstration of a
simple electrodiffusive pathway for spermidine efflux from cells.
Polyamines (spermine, spermidine, and putrescine) are present in almost all cells (Tabor and Tabor, 1984), and have important roles in stabilizing DNA. Recently, they have been implicated in physiological regulation of potassium channels, from inside cells (Lopatin et al., 1994; Ficker et al., 1994; Fakler et al., 1995), and of glutamate receptor channels from outside (Usherwood and Blagborough, 1994; Romano and Williams, 1994). Polyamines are taken up by, and released from, cells, but the mechanisms remain poorly understood (Khan et al., 1994). In general, polyamine influx is reported to be saturated at low micromolar concentrations (Seiler and Dezeure, 1990; Khan et al., 1991). Polyamine efflux has received less attention, although there is a general consensus that both influx and efflux depend on the membrane potential (Khan et al., 1994; Shaw, 1994). Polyamine transporters have been cloned from bacteria (Furuchi et al., 1991; Kashiwagi et al., 1993), but no evidence is available on the molecular nature of eukaryotic transport systems.
We recently
reported that polyamines cause inward rectification of potassium (Kir)
channels (Lopatin et al., 1994; Ficker et al., 1994;
Fakler et al., 1995), by steeply voltage-dependent block of
the channel pore. Kir channels are present in most cells, and
polyamine-induced rectification is likely to be ubiquitous. Detailed
biophysical examination of this process suggests that in blocking the
Kir channel, polyamines traverse a significant fraction of the membrane
electric field (Lopatin et al., 1995). It is possible that
polyamines might actually permeate the channel fully, but behave
macroscopically as channel blockers due to a long residence time within
the pore. Accordingly, we were prompted to examine the possibility that
efflux through Kir channels is a significant pathway for spermidine
efflux. The results suggest that this is not the case, but provide
significant information on the mechanism by which spermidine efflux
does occur. First, under voltage-clamp conditions, spermidine efflux is
strictly dependent on membrane potential, as expected for a trivalent
species. Second, voltage-dependent spermidine efflux is strongly
inhibited by Ba and Ca
, but not
tetraethylammonium ions or verapamil. We conclude that efflux of
trivalent spermidine occurs through an electrodiffusive pathway, and
hypothesize that the pathway is through cation channels in the cell
membrane. Such a pathway may be a universal route of polyamine flux in
different cells.
In voltage-clamp experiments, oocytes were incubated in 200 µl of solution. At the end of the test period, the solution in the chamber and 2 additional volumes of wash solution were collected, without disturbing the oocyte or the electrodes. Oocytes were washed with a further 3 volumes of the next test solution, before beginning the next test period.
Figure 1:
[H]Spermidine
release from Xenopus oocytes. A,
[
H]spermidine released into the medium (as a
percentage of the total injected into the oocyte) in 0.1, 1, and 18 h,
for control oocytes, and oocytes expressing HRK1 inward rectifier
K
channels. These experiments were performed in ND96
solution without Ca
. The error bars show
mean ± S.E. in this and other figures (n = 12
oocytes in each case). B, [
H]spermidine
released in 90 min from HRK1 expressing oocytes incubated in ND96 (ND), ND96+1 mM Ca
, or KD
solutions. C, elution profile of different polyamine species
(fluorescence), and of tritiated species released from oocytes during a
3-h incubation in KD98 solution. Oocytes were injected 24 h previously.
The same pattern of radioactivity was observed for oocytes injected 1 h
prior to release assay.
Figure 2:
Divalent cations inhibit
K stimulated [
H]spermidine
release. A, [
H]spermidine released in 90
min from control and HRK1 expressing oocytes incubated in KD98 solution
± 1 mM Ba
. Graph shows mean ±
S.E. for n = 7 oocytes in each case. B,
[
H]spermidine released in 90 min from HRK1
expressing oocytes incubated in KD98 solution plus 0-1 mM Ba
. Graph shows mean ± S.E. for n = 4-18 oocytes. C,
[
H]spermidine released in 90 min from HRK1
expressing oocytes incubated in KD98 (KD), or ND96 (ND), solutions in the absence of Ca
(pre-), after inclusion of 1 mM Ca
in the medium (+Ca), and after removal (post-) of Ca
from the incubation medium (n = 6 oocytes in each
case).
Figure 3:
In
the absence of divalent cations, [H]spermidine
release is by electrodiffusion. [
H]Spermidine
released in 120 min from HRK1 expressing oocytes versus
E
. E
was
calculated from the potassium reversal potential (E
). E
was varied by
changing [K
] in the incubation medium by
mixing KD98 and ND96 solutions. Results are shown for 3 oocytes for
which all solution changes were successfully completed. Solutions
changes were made in increasing concentration of potassium (
), or
in decreasing concentration of potassium (
,
). The dashed lines indicate the relationship predicted for simple
electrodiffusion of a species with 0, 1, 2, 3, or 4 charges according
to the GHK equation (see text).
where p = permeability, z =
charge of the permeating ion, X and X
are the internal and external concentrations of
the permeating ion (in this case X
= 0), E
is the membrane potential, and F, R, and T have their usual meanings.
Figure 4:
[H]Spermidine
release is not dependent on ion composition of the medium. Upper
panel, slow time base record of E
and membrane current, from an oocyte clamped at a holding
potential of -90 mV (left), then 0 mV (middle),
then -90 mV (right) for 30 min in each case. The oocyte
was bathed in ND96 solution. The top panel shows
[
H]spermidine released during each 30-min period.
During each period, the E
was ramped from
-90 to 0 mV every minute, and sample fast time base records of E
and membrane current are shown in the bottom panel, from the times indicated. B, results of
individual experiments (numbered) like those in A, for
voltage-clamp to -90, then 0, then -90 mV (oocytes
1-7), or to 0, -90, and 0 mV (oocytes 8-12).
Experiments in ND96 are shown as white boxes, experiments in
KD98 are shown by black boxes.
Figure 5:
Spermidine release under voltage-clamp is
by electrodiffusion. A, averaged results for
[H]spermidine released from voltage-clamped
oocytes, clamped to a control voltage (either -90 mV, or 0 mV),
then to a test voltage (either 0, -90, or +30 mV), then once
more at the control voltage. Results of experiments performed in ND96
and KD98 solutions are pooled. Graph shows mean ± S.E. for n = 7 (left panel), n = 6 (middle panel), and n = 4 oocytes (right
panel). B, all data from B are pooled and plotted versus voltage-clamp potential. Dashed lines indicate
the relationship predicted for simple electrodiffusion of a species
with 0, 1, 2, 3, or 4 charges according to the GHK equation (see
text).
Ca sensitivity of release is maintained under
voltage-clamp conditions (Fig. 6). Thus, depolarization (from
-90 to 0 mV) strongly stimulates release in the absence of
Ca
, but there is no stimulation in the presence of 1
mM Ca
. The background stimulation by
Ca
under hyperpolarized conditions in
nonvoltage-clamped oocytes (Fig. 1B) is also apparent
under voltage-clamp at -90 mV (Fig. 6).
Figure 6:
Calcium sensitivity of release is
maintained under voltage-clamp conditions.
[H]Spermidine released in 30 min from
voltage-clamped oocytes incubated in ND96, solution in the absence (left) or presence (right) of 1 mM Ca
. Oocytes were clamped to -90 mV for 30
min, then to 0 mV for 30 min, then to -90 mV once more for 30 min (n = 4 oocytes in each
case).
These results
show that a major pathway of spermidine efflux is through a simple
electrodiffusive pathway that is blockable by Ba and
Ca
ions, consistent with efflux through a membrane
cation channel. Many cation channels are also blocked by
tetraethylammonium (TEA) ions or verapamil (Hille, 1992), but as shown
in Fig. 7, neither agent blocked depolarization-induced
spermidine release, matching the pharmacological profile reported for
mechanosensitive cation channels in oocyte membranes (Yang and Sachs,
1989, 1990). We attempted to modulate mechanosensitive cation channel
activity by manipulating external osmolarity. Halving or doubling
osmolarity caused oocytes to swell, or shrink, respectively (not
shown), but failed to stimulate spermidine efflux or activate
significant currents.
Figure 7:
Spermidine release is insensitive to
TEA and verapamil. [
H]Spermidine
released in 90 min (left) or 30 min (right) from HRK1
expressing oocytes in KD98 solution ± 1 mM tetraethylammonium (TEA, left), and ± 10
µM verapamil (ver, right) (n = 8
oocytes in each case).
Although polyamine transport into and out of
cells has been studied for many years, molecular mechanisms remain
poorly understood (Khan et al., 1994). Cellular uptake has
received the most attention, and it is clear that there are high
affinity uptake systems with saturation at low micromolar levels of
polyamines in oocytes and other animal tissues (Khan et al.,
1990; Kano and Oka, 1976; Saunders et al., 1989; Gilad and
Gilad, 1991). There is some evidence for
``transacceleration'' of polyamine flux, whereby raising the
transmembrane polyamine concentration paradoxically increases flux from
the cis-side of the membrane, indicative of futile cycling of an
exchange process (Byers et al., 1990; Mackarel and Wallace,
1994). A literature survey suggests that little is known about the
mechanistic basis of polyamine efflux. One reasonably consistent
finding is that depolarization, or maneuvers likely to induce
depolarization (e.g. increased extracellular
[K], stimulation of depolarizing ion
currents, metabolic poisoning), tend to stimulate efflux, or to inhibit
influx (Kashiwagi et al., 1986; Khan et al., 1992;
Poulin et al., 1995; Fage et al., 1992, 1993; Nicolas et al., 1994). This finding prompted us to systematically
examine the hypothesis that spermidine efflux can occur by simple
electrodiffusion. The use of Xenopus oocytes provides three
essential technical advances for this purpose: 1) high level expression
of potassium channels permitted voltage-clamping by manipulation of
extracellular [K
]; 2) the large size of
oocytes permitted spermidine to be introduced by injection; 3) injected
oocytes could be voltage-clamped using a two-microelectrode
voltage-clamp. The results show for the first time that spermidine
efflux is strictly dependent on E
in the absence
of external divalent cations (Fig. 4). The results obtained
under two-microelectrode voltage-clamp demonstrate that the effect of
changing external [K
] is entirely a
consequence of the change in E
, and not of changes
in external [Na
], or
[K
] per se. Thus the results
provide direct evidence against a Na
or K
cotransport or countertransport mechanism being involved in
spermidine efflux under the conditions of our experiments.
Although
there is no a priori reason to suspect it, it might be argued
that depolarization of the oocyte introduces some nonspecific leakage
through which spermidine efflux is occurring. However, the specific
blockage of this effect by trans-Ba or Ca
ions argues strongly against this, and instead is consistent with
efflux occurring through a membrane pore. In normal high
[Na
] solutions, the oocyte is
hyperpolarized, and electrodiffusive release is slow. Under these
conditions, Ca
(1 mM) stimulates what
appears to be a voltage-independent efflux. Similar mixed
agonist/antagonist effects of divalent cations on polyamine uptake have
been reported in human breast cancer cells (Poulin et al.,
1995).
It is not yet clear which channels constitute the pathway for
spermidine efflux from oocytes. Mechanosensitive non-selective cation
channels are present in oocyte membranes at high density (Yang and
Sachs, 1990) and, like polyamine efflux, are inhibited by
Ca and Ba
, but not by
TEA
or verapamil (Yang and Sachs, 1989, 1990).
Attempts to manipulate the open probability of these channels in intact
oocytes were not successful, and indeed, we have found no reports of
successful manipulation of mechanosensitive channels in whole cell
currents in the literature. At present we may only speculate that the
pathway for electrodiffusive release of spermidine from oocytes, and
perhaps other cells, is through mechanosensitive, non-selective cation
channels.