Proton modulation of outward K+ currents in interferon-γ-activated microglia

ELSEVIER

Neuroscience Letters 206 (1996) 101-104

HSlltll I[II[RS

Proton modulation of outward K ÷ currents in interferon-v-activated microglia Claudia Eder*, Uwe Heinemann Abt. Neurophysiologie, InstitutJ~ir Physiologie der Charitd, Humboldt Universiti~t zu Berlin, Tucholsky Strasse 2, D-lOll7 Berlin, Germany Received 17 January 1996; revised version received 2 February 1996; accepted 2 February 1996

Abstract

Whole-cell outward potassium currents (IK) were measured in interferon (IFN)-y-activated cultured murine microglial cells. Acidification of the external milieu moved the threshold of activation of 1K in a depolarizing direction, while alkalinization showed the opposite effect. A shift of more than 20 mV of the steady-state activation and inactivation curves of 1K in hyperpolarizing direction was measured when pH was changed from 5.8 to 7.8. The time-dependent inactivation of / K was slower when superfusing cells with acid solutions than with alkaline ones. In contrast, variations in the pH of the intracellular solution did not alter kinetics of 1K. However, alkalinization of the internal solution from a pH of 5.8 to 7.8 led to a two-fold increase in the current density o f l K.

Keywords: Microglia; Potassium current; pH modulation; Acidosis; Alkalosis

Changes in the intracellular pH (pH i) have been observed in cells of the central nervous system after depolarization [2]. In neurons an intracellular acidification, often induced by calcium ion influx, was measured during the electrical activity [ii]. Moreover, at positive holding potentials pH i increases to a steady state that depends on the electrochemical gradient for protons across the neuronal cell membrane [16]. In contrast, astrocytes and oligodendrocytes undergo a rapid intracellular alkalinization when they are depolarized. This is probably caused by an influx of HCO 3- using a Na+/HCO3 - cotransporter [4,11]. Shifts in the extracellular pH (pHo) have also been observed in response to neuronal activity. A prolonged acidification of the extracellular space was detected upon stimulation in many neuronal preparations [3]. Such changes in pH o were more prominent during repetitive stimulation, epileptiform activity and spreading depression [3,13]. Upon extracellular alkalinization, a voltage-gated proton current can be induced in resting microglia, while the inward rectifying K ÷ current is not influenced by pHo shifts in these cells [6]. In contrast to these resting cells, * Corresponding author. T,~I.: +49 30 28026302; fax: +49 30 28026669.

cytokine-activated microglia mainly exhibit voltage-gated outward K ÷ currents [7]. In the present paper, the modulatory effects of pH i and pHo variations on the kinetics of K ÷ currents (IK) in interferon (IFN)-7-activated microglial cells are reported. Microglia were obtained from brain cell cultures of newborn ( B 1 0 × C3H/HeJ)F 1 mice which originated from a breeding stock supplied by Hamann-Winkelmann, Borchen, Germany. Mixed brain cell cultures were prepared as described previously [7]. After at least 10 days of incubation microglial cells were harvested by shaking the cultures (1 h, 300rpm) to detach weakly adherent cells from the astrocytic monolayer. Isolated microglia were propagated up to 8 days on glass coverslips in 24well Costar plates (5 x 104/1 ml) for patch clamp experiments. In order to activate microglia 10 U/ml IFN-y (Genzyme, Germany) were added for 24 h to the culture medium which results in the expression of outward K ÷ currents in the majority of microglial cells [8]. Membrane currents were measured in the whole-cell configuration of the patch clamp technique using a recording and analyzing system as described previously [6]. For current recordings the electrodes were filled with high K ÷ solutions containing (in mM): KCI, 120; CaCI 2, 1; MgCI 2, 2; HEPES, 10; EGTA, 11; D-glucose, 20. The pH

0304-3940/96/$12.00 © 199,5 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2433-0

102

C. Eder, U. Heinemann I Neuroscience Letters 206 (1996) 101-104

of the intracellular solution (pHi) was adjusted to 5.8, 7.3 or 7.8. The extracellular solutions contained (in mM): NaCI, 120; KCI, 5.4; CaCI2, 2; MgC12, 1; HEPES, 10; Dglucose, 25. The external superfusing solution was adjusted to a pH (pHo) of 5.8, 6.8, 7.4 or 7.8. All recordings were done at room temperature (20-23°C). A four-barrel microperfusion pipette was positioned at a distance of about 30-50ktm from the cell from which recordings were being made. Cells were superfused with external solutions of different pHo values. Data are presented as mean values -+ SD of the number of experiments indicated. Fig. 1 shows superimposed whole-cell IK of an example of IFN-),-activated microglial cells superfused with extracellular solutions of pH o 5.8, 6.8 or 7.8. The threshold of activation of I X was more positive at acid pH o than 200ms+30 -60 ~

A

PHo 5.8

~]11... i B

pH o 6.8

PHo7.8

_.LL

I

:

J"~

.

r.

I

D

_ _ l lOOpA 50ms ,I

1.0

~

0.8

~

0.4

/...!

;~ 0.2

."

0.13 -50 t

,

I

/t ,

I

,

f

,

-10 10 Membranepotential(mV) -30

30

Fig. 1. Steady-state activation behaviour of 1K at different pH o values. Voltage-gated outward K + currents were activated by depolarizing steps from a holding potential of -60 mV. Voltage pulses were applied for 200 ms to potentials between -60 and +30 mV in l0 mV increments. The pH i was fixed to a value of 7.3, pH o was adjusted either to 5.8 (A), 6.8 (B) or 7.8 (C). (D) Using a Boltzmann equation, steadystate activation curves of 1K were fitted after superfusion of cells with external solutions at pH 5.8 (dashed line), 6.8 (pointed line) and 7.8 (solid line).

at alkaline pHo. It was determined at about - 2 0 mV during superfusion with a solution at pHo 5.8, at about - 3 0 mV for a pH o of 6.8 and at about ---40 mV for a pH o of 7.8. During extracellular alkalinization the steady-state activation curve of I K shifted in a negative direction. The half-maximal activation of IK was determined to be -2.8 mV ( n = 8) at pH o 5.8. At pHo of 6.8 and of 7.8 the corresponding values were -15.3 mV ( n = 8) and -27.3 mV (n = 8), respectively. The shift of the activation curve was approximately parallel, i.e. the slope factor of the curve did not markedly change during variations of pHo. Moreover, during variations of the extracellular pH o from acid to more alkaline values, a shift in the steadystate inactivation curve of I K in a hyperpolarizing direction was observed (not shown). To study the inactivation behaviour the holding potential of the cells was varied between - 8 0 and 0 mV in 10 mV increments. Several minutes after establishing the actual holding potential, a test pulse to +30 mV was applied for 200 ms. Steady-state inactivation curves of I X were fitted by a Boltzmann equation. A half-maximal inactivation of IK was obtained for pH o 5.8 at -22.9 mV (k = --4.7 mV; n = 9), for pHo 6.8 at -33.9 mV (k = -6.0 mV; n = 9) and for pHo 7.8 at -45.8 mV (k = -4.9 mV; n = 9). The effect of variations of pH o on time-dependent inactivation of I X is illustrated in Fig. 2. During superfusion of microglial cells with external solutions at a pHo of either 5.8, 6.8 or 7.8, inactivation behaviour of I K differed dramatically. However, in all three cases the decay of Ix could be fitted by one exponent. The time constants were larger when IK was evoked by voltage commands near the activation threshold of Ix than those determined when I X was evoked by large depolarizing test pulses at which they reached finally a stationary value. The time course of inactivation of I K did not markedly change at potentials more positive than - 2 0 mV determined at pHo 7.8, while at pHo 5.8 the inactivation of I X could be described by nearly identical time constants only at test potentials more positive than +20 mV. The time-dependent inactivation of Ix which was evoked by a test pulse to +80 mV (for 2000 ms) is shown in Fig. 2D. I X decayed more slowly at acid pHo values than at alkaline ones. At a command pulse to + 8 0 m V mean time constants were 810.8_+ 46.2 ms (n = 6) for pH o 5.8, 592.0 _+33.7 ms (n = 8) for pH o 6.8, and 488.8 +_28.6 ms (n = 7) for pH o 7.8, respectively. To examine whether changes of pHi alter outward K ÷ currents in IFN-y-activated microglia, the cells were patch clamped twice using patch pipettes which contained intracellular solutions of different pH values. During current recordings cells were superfused with an extracellular solution with a stable pH o of 7.4. The pH of the intracellular solution was adjusted either to 5.8 or to 7.8. In order to minimize artefacts, half of the ceils analyzed were perfused first with the acid internal solution and then with the alkaline one. The other 50% of cells were patch-

103

C. Eder, U. Heinemann /Neuroscience Letters 206 (1996) 101-104

A

1

pHo 5.8

B

2000ms+80

pHo 6.8

r-

C

D

pHo 7.8

2000

!~,

~ 15o0

~ . ~

~1000

=

~

i~,

pHo 5.8

I oHo6

~

~oo

100pA 500ms

0 0 .... 5()0 10'00 16'00 ' 2000 Command pulse duration (ms)

Fig. 2. Time-dependent inamivation of IK at different pHo values. Voltage pulses from the holding potential (---60mV) to potentials between --60 and +80 mV were applied in 20 mV increments for 2000 ms duration. Examples of current recordings at pH 5.8 (A), 6.8 (B) and 7.8 (C). (D) Monoexponential fit of time course of inactivation for currents evoked by a pulse to +80 mV at different pHo as indicated. clamped using first the alkaline intracellular solution before perfusing cells with the acid solution. At a pH i of 7.8 the amplitudes of I K seemed to be larger than those measured at a pHi of 5.8 (Fig. 3). Due to possible changes in cell surface area after removal of the first patch pipette, current densities of IK were determined. In order to estimate current density the capacitance of the cells was determined and was used to obtain an estimate of cell surface area. The total cell capacitance ranged from 1035 pF for all microglial cells analyzed. The cell capacitance measured with the second patch pipette was I - 6 pF smaller than that measured with the first pipette, independently on the containing intracellular solutions. The cell surface area was estimated by assuming a specific membrane capacitance of 1/~F/cm 2, and the peak amplitudes of I K were related to the individually estimated cell

surface area. After depolarization to +30 mV, for both intracellular solutions tested the current density of the elicited IK of individual cells varied over a wide range: between 3.8 and 14.8/~A/cm 2 (n = 11) at pHi 5.8, and between 7.2 and 31.1/zA/cm 2 (n = 11) at pH i 7.8. Probably these variations in current density of I K from cell to cell were a result of the different activation state of the cells after cytokine treatment (Eder et al., in preparation). Therefore, the increase in current density of I K at pHi 5.8 and at pH i 7.8 was determined for each cell measured. During alkalinization of the intracellular solution from pH i 5:8 to 7.8 the current density of I~( increased by a factor of 1.92 _+0.29 (n = 11). In contrast to the current density, kinetics of I K did not change as a result of variations of pHi. Analyzing the threshold of activation, time-dependent inactivation,

200ms

A

,00L /

B

(--)

J50ms

100pA

~.

-50

-30

310 -10 10 Membranepotential (mY)

Fig. 3. Effect of pHi on/g. (A) 1Kevoked by a test pulse from -60 to +30 mV (200 ms) perfusing the cell first with a solution at pHi 7.8 and after that with a solution of pHi 5.8. (B) Current-voltage-relationshipof IK for the cell shown in (A) (closed symbols, pHi 7.8; open symbols, pHi 5.8).

104

C. Eder, U. Heinemann / Neuroscience Letters 206 (1996) 101-104

steady-state activation and inactivation curves, no differences were found between whole-cell outward K+ currents measured at pHi 5.8 and those measured at pHi 7.8 (not shown). Outward K+ currents of IFN-),-activated microglial cells were modulated by changes in PHo and pHi. Whereas extracellular acidification shifted both the steady-state activation and inactivation curves for I K in depolarizing direction in a nearly parallel fashion, extracellular alkalinization shifted them in a hyperpolarizing direction. In agreement with similar observations made at a variety of preparations [10,12] this effect can be explained by proton-induced modification of membrane surface potentials. However, since extracellular acidification slows inactivation of 1K, independent of voltage, a second mechanism of modulation of IK by protons is possible. A binding site for protons close to or directly on the K+ channel might modify its gating. The existence of such binding sites on the extracellular surface of voltagedependent K÷ channels has been postulated for divalent cations, which could modulate channel gating in a similar way as protons [14,17]. Moreover, protonation of an acid group within the pore could also be responsible for the effects described [9]. Intracellular acidosis reduces and alkalosis increases 1K in activated microglial cells without changing the activation and inactivation behaviour of the current, indicating the absence of effects on surface charges in the pH range studied. Thus, it is more likely that the ability of K÷ channels to open increases with intracellular alkalinization. The possibility of intracellular protons inhibiting K+ channels has been reported for the inward rectifier [6,19], the delayed rectifier [5,I5] and the Ca2+-activated K÷ channels [18]. We would like to thank A. Laf3 and S. Latta for the preparation of cell cultures and R. Guti&rez for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, project SFB 507/ C3. [1] Ahmed, Z. and Connor, J.A., Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurones, J. Gen. Physiol., 75 (1980) 403--426. [2] Chesler, M., The regulation and modulation of pH in the nervous system, Prog. Neurobiol., 34 (1990) 401-427.

[3] Chesler, M. and Kaila, K., Modulation of pH by neuronal activity, Trends Neurosci., 15 (1992) 396-402. [4] Deitmer, J.W. and Schlue, W.R., An inwardly directed, electrogenie sodium-bicarbonate cotransport in glial ceils of the leech central nervous system, J. Physiol., 411 (1989) 179-194. [5] Deutsch, C. and Lee, S.C., Modulation of K+ currents in human lymphocytes by pH, J. Physiol., 413 (1989) 399--413. [6] Eder, C., Fischer, H.G., Hadding, U. and Heinemann, U., Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor, Pfl0gers Arch., 430 (1995) 526-533. [7] Eder, C., Fischer, H.G., Hadding, U. and Heinemann, U., Properties of voltage-gated potassium currents of microglia differentiated with granulocyle/macrophage colony-stimulating factor, J. Membr. Biol., 147 (1995) 137-146. [8] Fischer, H.G., Eder, C., Hadding, U. and Heinemann, U., Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states, Neuroscience, 64 (1995) 183-191. [9] Hille, B., Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA, 1992, pp. 393-397. [10] Iijima, T., Ciani, S. and Hagiwara, S., Effects of the external pH on Ca channels: experimental studies and theoretical considerations using a two-site, two-ion model, Proc. Natl. Acad. Sci. USA, 83 (1986) 654-658. [111 Kettenmann, H. and Schlue, W.R., Intracellular pH regulation in cultured mouse oligodendrocytes, J. Physiol., 406 (1988) 147162. [12] K16ckner, U. and Isenberg, G., Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance, J. Gen. Physiol., 103 (1994) 665--678. [13] Kraig, R.P., Ferreira-Filho, C.R. and Thompson, P., Alkaline and acid transients in cerebellar microenvironment, J. Neurophysiol., 49 (1983) 831-850. [14] Mayer, M.L. and Sugiyama, K., A modulatory action of divalent cations on transient outward current in cultured rat sensory neurones, J. Physiol., 396 (1987) 417--433. [15] Moody, W.J. and Hagiwara, S., Block of inward rectification by intracellular H+ in immature oocytes of the starfish Mediaster aequalis, J. Gen. Physiol., 79 (1982) 115-130. [16] Preisig, P.A. and Alpern, R.J., Contributions of cellular leak pathways to net NaHCO 3 and NaC1 absorption, J. Clin. Invest., 63 (1989) 1859-1867. [17] Spires, S. and Begenisich, T., Chemical properties of the divalent cation binding site on potassium channels, J. Gen. Physiol., I00 (1992) 181-193. [18] Stampe, P. and Vestergaard-Bogind, B., The Ca2+-sensitive K+conductance of the human red cell membrane is strongly dependent on cellular pH, Biochim. Biophys. Acta, 815 (1985) 313-321. [19] Wanke, E., Carbone, E. and Testa, P.L., K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane, Biophys. J., 26 (1979) 319324.