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1 From the Department of Neurophysiology, Paul Flechsig Institute of Brain Research, University of Leipzig, D–04109 Leipzig; and the 2 Department of Ophthalmology, Eye Hospital, University of Leipzig, D–04103 Leipzig, FRG.
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Abstract |
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METHODS. Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients. The whole-cell and the cell-attached configurations of the patch-clamp technique were used to characterize the current densities of different K+ channel types and the activity of single Ca2+-activated K+ channels of big conductance (BK).
RESULTS. Cells from patients displayed a less negative mean membrane potential (-52.8 mV) than cells from healthy donors (-80.6 mV). However, the membrane potentials in cells from patients scattered largely between -6 and -99 mV. The inwardly rectifying K+ permeability in cells from patients was strongly reduced (0.3 pA/pF) when compared with cells from healthy donors (6.0 pA/pF). At the resting membrane potential, single BK channels displayed a higher mean activity (open probability, Po, and channel current amplitude) in cells from patients (Po: 0.30) than in cells from healthy donors (Po: 0.03). The variations of BK current amplitudes were correlated with the variations of the membrane potential.
CONCLUSIONS. The dominant expression of inwardly rectifying channels in cells from healthy donors is thought to support important glial cell functions such as the spatial buffering of extracellular K+. The downregulation of these channels and the less negative mean membrane potential in cells from patients should impair spatial buffering currents and neurotransmitter clearance. The increased activity of BK channels may support the proliferative activity of gliotic cells via feedback regulation of Ca2+ entry and membrane potential.
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Introduction |
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The expression of ion channels by Müller cells may change in response to various conditions. Human Müller cells derived from patients with different eye diseases lack KIR currents, or express this current type at greatly reduced densities.8 The lack of KIR channels is accompanied by a shift of Müller cell’s membrane potential toward more depolarized potentials. During postnatal development of radial glial (Müller) cells of the rabbit retina, the expression of KIR channels increases strongly during the second postnatal week.9 In the first postnatal week, the amplitude of KIR currents is very small, whereas depolarization-activated outwardly rectifying K+ currents (particularly KA and BK currents) predominate the K+ permeability of the membrane. The resting membrane potential increases from low neonatal values (-40 mV) to high values negative to -80 mV at day 9. This shift of the membrane potential is accompanied by a correlated strong developmental decrease in the activity of BK channels as recorded in on-cell patches at the resting membrane potential.9
The aim of the present study was to evaluate whether the BK channel activity differs among human Müller cells freshly isolated from retinas of healthy donors and of patients with proliferative vitreoretinopathy (PVR). Because cells from patients display a less negative mean membrane potential than cells from healthy donors,8 and because BK channels are depolarization-activated,7 one should assume that the mean "native" (i.e., measured at the resting potential) open probability of BK channels is elevated under pathologic conditions. In addition, pathologic alterations in the activity of the other K+ channels were investigated to establish a "map" of the K+ current pattern characteristic for "reactive" human Müller cells in PVR.
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Methods |
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Electrophysiological Recordings
The whole-cell and the cell-attached configurations of the
patch-clamp technique were used.10
The recordings were
made at room temperature. The bath solution contained (in millimoles)
110 NaCl, 3 KCl, 2 CaCl2, 1
MgCl2, 10 HEPES, and 11 glucose (pH 7.4 adjusted
with Tris-base). The pipette solution was made of (in millimoles) 10
NaCl, 130 KCl, 1 CaCl2, 2
MgCl2, 10 EGTA, and 10 HEPES (pH 7.2), resulting
in a free [Ca2+] of 26 nM. Gigaseals (5–10
G
) were formed with borosilicate pipettes (GB150F8P, Biological,
Science Products, Frankfurt/Main, Germany) that displayed
resistances between 5 and 10 M. Recordings were made using an EPC 7
amplifier (List Electronics, Darmstadt, Germany) and the TIDA 5.72
computer program (HEKA elektronik, Lambrecht, Germany). High
frequencies >1 kHz were cut off. The series resistance was compensated
by 30% to 60%.
Cell-attached patches were voltage-clamped at a pipette potential of 0 mV (i.e., at the native resting membrane potential), and de- and hyperpolarizing voltage steps of 1-seconds’ duration were applied. The pipette tips were placed onto the soma or the lateral face of the end foot. No significant differences in the single-channel activity were found between patches at the two different locations. Only those patches were used for evaluation of channel activity that contained active BK channels when the patch potential was stepped up to ±80 mV away from the resting potential.
During whole-cell recordings, the cells were voltage-clamped at -80 mV, and voltage steps from -160 to +200 mV were applied with an increment of 20 mV. The KA amplitude was determined as the peak current evoked by a voltage step to +10 mV after a 500–msec pre-pulse to -120 mV, subtracted from the current evoked after a 500–msec pre-pulse to -40 mV. The recordings of KA currents were performed in control solution as well as in extracellular solution containing 5 mM 4-aminopyridine (4AP); the two recordings were then subtracted to obtain the 4AP-sensitive transient currents. The whole-cell BK currents were measured between voltage steps to +160 and +200 mV, when the KIR currents were blocked by Ba2+ (1 mM). The membrane capacitance of the cells was measured in whole-cell recordings at the uncompensated capacitive artifact evoked by a hyperpolarizing voltage step from -80 to -90 mV, when the K+ currents were blocked by Ba2+ (1 mM).
Data Analysis
For whole-cell data, the membrane (holding) potentials are given
with respect to the intracellular side of the membrane. In
cell-attached data, the pipette potential means the voltage applied to
the pipette (extracellular side of the patch). Amplitude histograms of
the single-channel currents were used to calculate the open probability
(Po) as described
earlier.11
Po means the open
probability of a single K+ channel. Whole-cell BK
channel–mediated and KDR-mediated currents were
measured as steady-state currents in voltage-step traces that were
obtained during external application of Ba2+ (1
mM) to block KIR currents.
KDR currents were measured at steps between -20
and +20 mV. The currents and membrane potentials of the whole-cell
recordings were corrected both for the shunt through the seal and for
the junction potential. Statistical analysis (unpaired Student’s
t-test, Mann–Whitney U test, regression
analysis) and curve fits were made using the Graphpad Prism program
(Graphpad Software, San Diego, CA). Data are expressed as mean ±
SD.
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Results |
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The membrane potentials were measured as zero current potentials in I–V curves of the whole-cell currents. Examples of whole-cell recordings are shown in Fig. 1A . In Figure 1B , the density of the inwardly directed whole-cell conductance is plotted against the membrane potential for two populations of Müller cells: cells obtained from healthy donors and from patients, respectively. Although the membrane potentials of cells from patients were scattered over a wide range between -99 and -6 mV (mean -52.8 ± 20.8 mV, n = 158), the potentials of most cells from healthy donors were found within a relatively small range close to -80 mV (mean -80.6 ± 9.0 mV, n = 134). Exposure to extracellular Ba2+ ions depolarizes Müller cells, by blocking their KIR channels.6 12 As shown in Figure 1C , the extracellular application of Ba2+ (1 mM) decreased the mean membrane potential in cells from healthy donors and patients to similar "minimum" values of -38.7 ± 19.4 mV (n = 79) and -34.4 ± 20.9 mV (n = 45), respectively.
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Current density values from all cells tested are shown in Figure 1B . Although the inwardly directed currents in cells from healthy donors generally exceeded 1.5 pA/pF (mean 6.3 ± 2.8 pA/pF, n = 115, measured at voltage steps from -80 to -120 mV) they were mostly decreased below this "limit" in Müller cells from patients (mean 0.5 ± 0.6 pA/pF, n = 158, P < 0.0001). Extracellular application of Ba2+ (1 mM) strongly reduced the amplitude of the inwardly directed currents in cells from healthy donors to 0.3 ± 0.2 pA/pF (n = 76; Figs. 1D and 2 A, 2B). Because these currents were very small in cells from patients already in control solutions, their Ba2+-induced reduction was less dramatic, although the Ba2+-insensitive "remnant currents" were similar (0.2 ± 0.2 pA/pF, n = 43; Figs. 1D and 2C ). Because the Ba2+-sensitive currents are mainly mediated by KIR channels,6 12 these results indicate that the mean KIR current density in cells from patients was only approximately 5% of that in cells from healthy donors.
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(n = 115), the membrane resistance of cells from
diseased donors was 3.2 G (n = 161,
P < 0.0001). The latter value was not significantly
different from the values found in another series of experiments,
involving a block of the KIR currents by
extracellular application of 1 mM Ba2+, in cells
from both healthy (3.6 G, n = 77) and diseased (3.6
G, n = 45) donors.
KIR Channels
In on-cell recordings using 130 mM KCl in the pipette solution and
3 mM KCl in the bathing solution, inwardly directed currents through
single K+ channels of different types were
recordable at a pipette potential of 0 mV (i.e., the native resting
membrane potential). The most abundant channel type in membranes of
Müller cells from healthy donors was the inwardly rectifying
K+ (KIR) channel (Fig. 3)
. The KIR channels exerted a high open
probability (Po) >0.8 at the resting
potential (Fig. 3A)
. The Po of
KIR channels was barely voltage-dependent, with
high values recorded over a relatively wide voltage range around the
resting potential, and with slight Po
reductions when the membrane was strongly hyperpolarized. The open
channel I–V relationship of KIR
channels was linear, with a mean slope conductance of 21.7 ± 2.9
pS (Fig. 3B)
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Examples of single-channel recordings are shown in Figure 5A . The recordings were made at a pipette potential of 0 mV (i.e., at the resting membrane potential). Downward deflections indicate cation fluxes from the extra- to the intracellular side of the membrane through single K+ channels. In the records on cells from healthy donors, small deflections represent the activity of several KIR channels, which caused the noisy baselines of the records. Large deflections represent the activity of BK channels. At the resting potential, BK channels in cells from diseased retinas generally showed a lower mean current amplitude and a greater mean Po than channels recorded in cells from healthy donors, although there was a large variability among the individual data. The mean Po was 0.30 ± 0.26 for cells from diseased retinas (n = 58) and 0.03 ± 0.03 for cells from healthy retinas (n = 21; P < 0.0001). The mean amplitude of currents through single BK channels was -5.82 ± 2.30 pA for cells from diseased retinas (n = 59) and -9.06 ± 1.90 pA for cells from healthy donors (n = 21; P < 0.0001).
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Figures 5B and 5C illustrate the relationships between BK current amplitude and Po, respectively (both measured in on-cell patches at the resting membrane potential), and the entry potentials of the cells. The entry potential was measured in the current-clamp mode immediately after establishing the whole-cell configuration, when the single-channel recordings were finished. The entry potentials differed considerably among the individual cells. However, the mean entry potential of cells from healthy donors was found to be higher (-52.1 ± 15.0 mV, n = 13) than that of cells from patients (-25.4 ± 16.9 mV, n = 29; P < 0.0001). Significant correlations existed between the BK channel current amplitude and the entry potential, in cells from healthy donors and patients alike (Fig. 5B) . No correlations were found between the entry potentials and Po (Fig. 5C) .
The above-mentioned differences of the BK channel activity at the resting membrane potential (Fig. 5A) were accompanied by different activation and I–V curves. Figure 6A illustrates examples of on-cell channel recordings at different holding potentials. In the recordings from the cell of a healthy donor (left column), two types of channel openings are discernible. At positive pipette potentials, the noisy baselines of the traces reflect the activity of several KIR channels. At negative pipette potentials (i.e., when the membrane patch was depolarized), the activity of a single BK channel is visible. The currents through this channel reversed at a pipette potential of about -65 mV. In the recordings from the cell of a patient (right column), the activity of a BK channel prevailed at both negative and (although with smaller Po) positive pipette potentials. The channel currents reversed at a pipette potential of about -40 mV. Mean Po–voltage (i.e., activation) and I–V curves of BK channels in both populations of cells are shown in Figures 6B and 6C , respectively. The mean activation curve of BK channels in cells from patients is shifted by 50.4 mV toward more hyperpolarized membrane potentials (i.e., positive pipette potentials), compared with the cells from healthy donors (P = 0.0069; Fig. 6B ). Moreover, also the mean I–V curve was found to be shifted toward more negative membrane voltages in cells from patients (Fig. 6C) . The mean reversal potentials of the single channel currents were found at pipette potentials of -76.7 ± 15.1 and -48.0 ± 12.7 mV in cells from healthy donors (n = 6) and patients (n = 21), respectively (P = 0.0002). These values are very similar to the mean native membrane potentials of both cell populations mentioned above (Fig. 1C) . The slopes of the I–V curves, however, were not significantly different; the BK channels displayed mean slope conductances of 113.2 ± 18.9 and 120.6 ± 26.4 pS in cells from healthy donors and patients, respectively.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
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The membrane potential differences between cells from healthy donors
and from patients are assumed to cause much of the differences in the
mean BK channel activity of the two populations of cells; BK channels
of cells from patients displayed a higher mean
Po (Fig. 5A)
and (at physiological
K+ gradients, see above and Fig. 6C
) also larger
mean amplitudes of single channel currents. First, the individually
different entry potentials were significantly correlated with the
varying BK channel current amplitudes (Fig. 5B) . Second, the difference
between the BK channel current amplitudes of control and reactive cells
(3.2 pA at pipette potentials of 0 mV) can be explained by a difference
in the mean membrane potential of approximately 28 mV, according to the
slope conductance of the BK channels (Fig. 6C)
. Indeed, this value is
very close to the measured difference (
V 27.8 mV; Fig. 1C
).
On the other hand, there was no significant correlation between the Po of BK channels and the entry potentials of individual cells (Fig. 5C) , whereas the mean Po values showed a clear-cut voltage dependence, different for normal and reactive cells (Fig. 6B) . It is also noteworthy that the mean activation (Po–V) curve of BK channels in reactive cells was displaced toward positive pipette potentials much more (by 50.4 mV; Fig. 6B ) than the mean I–V curve (by 28.7 mV; Fig. 6C ). This suggests the presence of additional factors that increase the Po of BK channels in gliotic cells. It is well known that the Po of BK channels depends on several factors, including the intracellular [Ca2+]. It is feasible that in cells from patients the intracellular [Ca2+] may be elevated (e.g., via voltage-insensitive, nonspecific cation channels15 or Ca2+ channels,16 which both are activated by growth factors such as the basic fibroblast growth factor).15 16 Moreover, a direct modification of channel properties, for example, mediated by channel phosphorylation,17 or channel stimulation via other second messengers like arachidonic acid18 cannot be ruled out. Anyhow, whereas we have presently no indications for any upregulation of BK channel expression in reactive Müller cells, BK channel–mediated currents are dramatically increased in such cells, mainly but not solely due to their decreased transmembrane potential.
Regulation of K+ Channels in Reactive Müller
Cells
In contrast to the case of BK channels, there is evidence that
KIR channels are indeed downregulated in reactive
Müller cells, due to a decreased genetic expression, a decreased
insertion into the membrane, or a functional inactivation. For
instance, the low KIR channel–mediated current
density (Fig. 1D)
is certainly not caused by an exhaustion of cellular
energy stores. Kir4.1 channels, the predominant
KIR channel type of rabbit Müller
cells,3
have a Walker-type A ATP-binding domain in the C
terminus,19
and KIR currents of
human and monkey Müller cells were previously found to be
decreased by internal ATP depletion.20
However, when 1 mM
Mg–ATP was intracellularly applied with the pipette solution to
Müller cells from patients for up to 1 hour
(n = 3, data not shown) no increase of the
KIR currents was observed.
As of this time, there is poor knowledge about the regulation of K+ channels in Müller cells. It has been speculated that neuronal activity–induced increases of the extracellular [K+] may stimulate the insertion of K+ channels into the Müller cell membrane.21 In cultured Müller cells, the presence of the extracellular matrix protein laminin and insulin was recently shown to induce the expression and membrane insertion of KIR channels.3 In the degenerating retina of PVR patients, Müller cells may lose their contacts to "healthy," normally functioning neurons as well as to the laminin-containing basal lamina. This may induce a downregulation of KIR channels. Although this problem is far from being solved, it is noteworthy that similar reductions of the resting membrane permeability for K+ have been observed in astrocytes of epileptic foci,22 astrocytic tumor cells,23 and cultured astrocytes from mechanically induced glial scars.24 Furthermore, the downregulation of KIR channels in proliferating astrocytes23 24 suggests that there is a relation between cell proliferation and low K+ permeability of the membrane.
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Conclusions |
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Footnotes |
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Submitted for publication April 22, 1999; accepted June 30, 1999.
Commercial relationships policy: N.
Corresponding author: Andreas Bringmann, University of Leipzig, Paul Flechsig Institute of Brain Research, Department of Neurophysiology, Jahnallee 59, D–04109 Leipzig, FRG. E-mail: bria{at}server3.medizin.uni-leipzig.de
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