Expression of an outwardly rectifying K+ channel in rat microglia cultivated on teflon

Neuroscience Leuers, 160 (1993) 69-72 C 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

69

NSL09807

Expression of an outwardly rectifying K+ channel in rat microglia cultivated on teflon Wolfgang Norenberga , Kurt Appel b , Joachim Bauerb , Peter J, Gebicke-Haerterb and Peter Illes a "Department 0/ Pharmacology and hDepartment 0/ Psychiatry. University 0/ Freiburg, Freiburg ( FRG) (Received 3 March 1993; Revised version received 14 June 1993; Accepted 14 June 1993)

Key words:

Microglia; Monocyte; Potassium channel; Lipopolysaccharide; Teflon bag; Patch clamp; Polymerase chain reaction

Membrane currents of cultured human monocytes and rat microglia were recorded with the whole-cell patch clamp technique. Freshly isolated Inonocytes or resting (proliferating) microglia express only inwardly rectifying K+ channels. However, incubation in teflon bags leads to the expression of additional, outwardly rectifying K+ channels. The outward K+ conductance of microglial cells was inhibited by intracellular Cs' and extracellular 4-aminopyridine or tetraethylammonium. Functional similarities with the microglial outwardly rectifying K+ channel were found in the K.-channel oflymphocytes which has recently been cloned (RGK5). The polymerase chain reaction (PCR) was used to demonstrate the presence of RGK5-like mRNA in microglia.

Macrophages exhibit a number of voltage-dependent K+ currents [8]. These cells may be activated by various stimUli, such as interferon-y (lNFy) and bacterial lipopolysaccharide (LPS) [1]. Monocytes have to reach a certain stage of differentiation into macrophages (e.g. by CUlturing in teflon bags) before they are able to fully reSPond to INFy [2]. Microglia are monocyte-derived. resident macrophages of the brain [16]. The predominant K + Conductance in resting (proliferating) rat microglia [10] and human blood macrophages [12] is inwardly rectifying. LPS has been shown to induce an outwardly rectifying potassium conductance in both cell types [12. 13]. The aim of the present study was twofold. Firstly. to investigate whether culturing of these cells in teflon bags leads to the expression of an outward K + conductance similar to that induced by LPS. Secondly. to test by PCR Whether this channel is similar to a K+ channel (RGK5 [6] or according to a new nomenclature K v 1.3 [4]) cloned from peripheral immunocytes. Pure microglial cultures were prepared from newborn Wistar rats [9]. A more than 90% monocytic population Was obtained from lymphocyte-enriched preparations of Whole blood (buffy coats) of healthy donors [2, 3]. Monocytes or microglia were transferred for further cultivation into teflon bags (Biofolie 25. Heraeus, Hanau, Correspondence: P. Illes, Department of Pharmacology, University of Freiburg, Hermann-Herder-Strasse 5, 0-79104 Freiburg, FRG. Fax: (49) (761) 203-4235.

FRG). Both cell types were maintained in adherence culture for 1 day before use for electrophysiological experiments. irrespective of whether they were kept in suspension culture (teflon bags) or not. Whole-cell membrane currents were recorded with the patch clamp technique as described previously [13]. The bath solution contained (mM): NaCl. 160; KCI. 4.5; MgCI 2, 1; CaCI 2• 2; HEPES. 5; glucose. 11; pH 7.4 with NaOH. The pipette solution contained (mM): KCI, 150; MgCI2 , 2; CaCI2 , 1; EGTA. 11 (free Ca2 +, 0.01 ,uM); HEPES, 10; pH 7.3 with KOH. Voltage steps (100-300 ms) were given in 10 mV increments from a holding potential of -70 m V every 8 s. Pulse range was from -170 to +70 mY. For statistical evaluation inward currents evoked by steps to -170 m V, and outward currents evoked by steps to +30 mV were chosen. The percent changes of these amplitudes by various ions and compounds were calculated. Means ± S.E.M. of n trials are shown. The Student's paired I-test was used. One ,ug of total RNA extracted from microglial cells [5] was reverse transcribed for 2 h and eDNA was specifically amplified by addition of Taq DNA polymerase (Perkin Elmer. Uberlingen, FRG) and the following oligonucleotide primers: 5'-AATGAGTACTTCTTCGACCGCAACAGACCCAGCTTCGA and 5'-CCAATGAAAAGGAAGAAAATGAGCAGCCCCAG. corresponding to nucleotides 283-320 and 1042-1073 of the K + channel RGK5 cDNA sequence, respectively [6]. Polymerase chain reaction (PCR) conditions were as follows: I

70

min 94°C, I min 64°C, 1.5 min noe (10 cycles); I min 94°C, 1 min 62°C, 1.5 min noe (30 cycles). A 790 bp amplification product was purified from agarose gel after electrophoresis and partially sequenced. Hyperpolarizing voltage pulses generated small inward currents in human monocytes kept for 1 day in adherence culture, while depolarizing pulses had no effect (Fig. lAa). For steps beyond -130 mV, the current amplitudes decreased with time. In monocytes kept for 7 days in suspension culture, the capacitive transients became larger, probably indicating increased capacitance due to cell growth (Fig. lAb; see also ref. 12). The peak inward current had a higher amplitude than in monocytes cultured only for I day (Figs. lAb and 2A). Additionally, these cells developed an outward conductance which activated below 0 mV and was characterized by an oscillatory behaviour (Figs. lAb and 2A). Neither the inward nor the outward current inactivated during 100 ms voltage steps (Fig. lAb). Eighty-one out of 87 microglial cells grown for I day

in an adherence culture (93%) exhibited only inward currents in response to hyperpolarizing pulses (see also ref. 10); depolarizing pulses were without effect (Fig. I Ba). The inward currents exhibited prominent time- and voltage-dependent inactivation. When the cells were grown for 7 days in adherence culture, peak inward currents became larger (Fig. I Ba inset and Fig. 2C), but no outward currents could be elicited upon depolarizing steps. After 7 days in suspension culture, the amplitUde of the inward currents was the same as in I day old adherence cultures, but depolarizing steps below - 50 mV evoked an additional, slowly inactivating outward current in 24 out of 29 cells (83%) (Figs. IBb and 2B). The two conductances expressed in microglial cells grown in suspension culture appear to be K + selective. An elevation of the K+ concentration in the bath mediuI1l from 4.5 to 50 mMshifted the zero current potential of the 1- V curve to the right (-71.3 ± 3.8 mV at 4.5 mM K+ 2+ and -40.8 ± 4.3 mVat 50 mM K+; n =4; P < 0.01). Ba (5 mM) selectively inhibited the inward (70.7 ± 3.1 %;

Ab

Aa

Suspension (7 d) + adherence (1 d)

Human monocyte

Adherence (1 d) 4

i

P

~250PA

Bb

Ba

Suspension (7 d) + adherence (1 d)

20 ms

Adherence (7 d)

Rat microglia Adherence (1 d)

F!~500 20 ms pA

~250PA 50 ms

Fig. I. Induction of an outward current by culturing of human blood monocytes and rat microglia in teflon bags for 7 days. Voltage pulses were applied every 8 s from a holding potential of -70 mV in 10m V increments. Step range was from -170 to +70 mV. A: monocytes cultured in adherence culture for I day (Aa) or in suspension culture (teflon bags) for 7 days with 1 day of subsequent adherence (Ab). B: rat microglia cultured in adherence culture for I day (Ba) or in suspension culture for 7 days with I day of subsequent adherence (Bb). Inset: rat microglia cultured in adherence culture for 7 days. Current responses to every second voltage step are shown (Aa and Ba). or current responses to every second depolarizing voltage step, and to a hyperpolarizing step to -170 mV (Ab and Bb).

71

P < 0.01; n

A Human monocytes

500

[pAl

=

[mV] 100

-800

B

Rat microglia [mV]

500

.e

tttn'f! 100

c

Rat microglia

-800

[pAl

500

[pAl

100

-1500 Fig. 2. Current-voltage (I-V) relations of human monocytes and rat tnicroglia. Same stimulation conditions as in Fig. I. A: monocytes cultured in adherence culture for I day (0; II = 8) or in suspension culture (tefton bags) for 7 days with I day of subsequent adherence (e; II = 9). 8: rat microglia cultured in adherence culture for I day (0; II 8) or in suspension culture for 7 days with I day of subsequent adherence (e; /I ::: 8). C: rat microglia cultured in adherence culture for I day (e; II 9) or 7 days (e; II 9). Means ± S.E.M. from II cells are shown.

=

=

=

4 each) conductances. Finally, a substitution of the intracellular K + (1 SO mM) with an equimolar quantity of Cs+ blocked the outward conductance, but inhibited the inward conductance only at strongly hyperpolarized potentials (n S; not shown). The inwardly rectifying K+ current observed in 1 day old adherence cultures of human monocytes was shown to occur also in mature macrophages [11]. A similar K+ conductance was described both in resting [10] and LPSactivated rat microglia [13]. Culturing of monocytes and microglia in suspension culture results in the appearence of an additional outwardly rectifying K+ current, similar to that expressed after LPS-treatment [12, 13]. The outwardly rectifying microglial K+ channels resembled K+ channels of T- and B-lymphocytes (Kn) [8], which have recently been cloned (RGKS) [6]. In order to demonstrate the expression of Kn-like channels in monocyte-related microglia, oligonucleotide primers for two higly conserved regions of the RGKS and closely related sequences (e.g. DRKI [7], RCKI [14], KVl and 2 [IS]) were used in PCR amplifications. Since treatment with LPS apparently induced the same outward current as culturing of microglia in teflon bags, we have searched for channel mRNAs in both resting and LPS- (100 nglml for 12-24 h) treated microglia. Only one DNA band was obtained after electrophoretic separation of the amplification product both in untreated and LPS-treated microglia. The size of this band was 790 base pairs (bp), as predicted (Fig. 3A). Partial sequencing data of the purified PCR product (14S bp and 128 bp from forward and reverse primers, respectively) reveal absolute identity of these regions with the published sequence (Fig. 3B). The appearance of a single amplified eDNA (Fig. 3A) suggests that only RGKS or a closely related outwardly rectifying K+ channel is expressed in microglial cells. Our failure to demonstrate differences in expression of channel mRNA between resting and LPS-treated cells should not be overinterpreted. As mentioned above, 7% of the LPS-free microglial population did express outwardly rectifying channels. Taking into account the amplification efficiency of PCR, a distinction between 10 or IS times more or less mRNA in the original RNA preparation needs more sophisticated methodology. Alternatively, it is feasible that the channel gene is constitutively transcribed and LPS (or culturing in teflon bags) controls the synthesis of the channel protein or its insertion into the plasma membrane.

=

p < 0.01), but did not alter the outward conductance (3.4 ± 3.4%; P > 0.05; n =5 each). 4-Aminopyridine (1 mM) had the reverse selectivity (inward current, 0.9 ± 3.8% inhibition, P> 0.05; outward current, 87.7 ± 2.S% inhibition, P < 0,01, n =4 each). Tetraethylammonium (10 mM) depressed both the inward (71.5 ± 7.8%; P < 0.01) and outward (S9.3 ± 11.9%;

This work was supported by grants of the Deutsche Forschungsgemeinschaft to P.I. (SFB 32S) and P.J.G.-H. (Ge 486/6-1).

72

B

A

5' - prim r

3' - prim e r

ACCT

ACCT

10 33 6 53

79 0 bp

+ LP S

RT

M

(bp)

T A nl C 38 3 - A-

T A C nt A - 9 73

Fig. J. Po l ym er~se chai n reac ti on (P R) amplifica ti o n and p~ rt ia l nucleotid e sequence of th e o utwardl y rectifyin g K ' channel of microgli al cells, A: gelelectrophoretic se para li o n of P R product amplificd from I Ji g of mi croglia l R NA usin g K' chann el RG KS-specifi c primers. Molec ul ar size mar kers (M) are indica led . Onl y a si ngle D NA ba nd cOl1l ai nin g 790 base pai rs (bp) was o bla ined bOlh in untrea ted and lipopolysaccharid e- (LPS: 100 nglml) Irea ted mi crogli a. Amplified cD NA did not o ri ginate fro m ge nomi c DNA . sin ce sa mpl es no t treated with reve rse Iranscript ase (RT) did nol produce a PR product. B: parli al nucleolide (n t) sequence of790 bp PC R prod uct. Left side. seq uence obtai ned fro m fo rwa rd (5') primer: right side. sequ ence ob ta ined from reve rse (:1') primer.

Ada ms. D.O. and Il am il lO n, T.A., Molec ul ar transducti onalmc han isms by which IFNy and o ther signa ls reg ul ale macro ph age develo pme nl , Immunol. Rev., 97 (1 987) 5 n . 2 And reesen. R .. Ga dd . .. Brugger. W.. Ui hr. G .W. ,lIld Atkin s. R. ., Acti va li n of hu ma n mo nocy tc-deri ved mac ro phages cul lu red on tefl on : res ponse 10 inlerferon -y dU ri ng termi nal ll1a lu rali on In vil ro, Imm uno blology, 177 ( 1988 ) 186 198 . J Bauer, J., Ga nt er. U .. Geige r. T. . Jaco bshagc n. U .. Hirano, T .. Matsud a. T .. Kl shlm olO, T. , And us. T., Ac . G .. Gem /.. . W.
10

II

12

13

1-1

15

16

all ow grow th and iso lali o n of mi crogli al cells. J. Nc urosci.• 9 ( 1989) 183 194. Kell enm ann . H .. Il o ppe. D .. Go ttm ann . K .. Banati . R. and Kn! uI Lberg, ulturcd microgli al cells ha ve a di slincl pallern of membrane chann els differenl rro m pcrit onea lm acro phages. J. Neurosel. Res .. 26 ( 1990) 278 287. Nelso n. D.J .• .l ow. B. and .l ow. F.. Wh ole-ce ll currents in macrophages: I. Hum an monocYle-derived macrophages. J. Membrane Bioi., 11 7 ( 1990) 29 44 . Nelso n. D.J .. .l ow. B. and .l ow. F.. Lipopolysaccharid e inducli on of o ut ward pO l a~siull1 currenl ex pressio n In hum an monocy te-deri ved nHlcroph ages: lac k of co rre latio n with secreti o n. J. Membrane BiOI.. 1:!5 ( 1992) 207 218. N,ire nbe rg. W.. Ge bicke-Il ae n er. P.J . and Ill es. 1'.• Inn ammalOr) , lImull Induce a new K ' o Ul ward current in cult ured ra l microglia . Ne urnsc l. Letl. , 147 (1 992 ) 171 174. Stii hme r. W., loc ker. M .. Sa km ann. B.. Seebu rg. P.. Baum ann. A.. ,rupe. A. and Po ngs. 0 .. POla'os ium chann els ex pressed from ral brain eD NA have del ayed recllfier pro perti es. FE BS Lett .• 242 ( 1988) 199 206. Swam,on. R .. M ar~ h a l l. J .. Smilh . .I ,S .. Will ia ms. .I .B.. Boyle, M.B.. Fo lander. K . I uneau. c..I .. Antan ovagc. J.. Oli va , .. Buhro\v. .A . Benn ell . .. Slei n, R.n . a nd KaCl marek. LK .. lo nin g and ex pression of cD NA ,lIld gc nonll\: clone cncodlll g three dela yctl rec lifie r po ta'S llIlll chann els in ral brain . Ne uro n. 4 ( 1990 ) 929 939. 1 heek. D.P. an d Slrell . W.J.. A Chl'(1I1 1cie of l11icrog lia l o nlOgcn}. G il a. 7 (1 99.1) 5 R.