Ca2+-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers

Diabetologia, Jul 1992

Summary This study evaluates the use of the planar lipid bilayer as a functional assay of Ca2+-activated K+ channel activity for use in purification of the channel protein. Ca2+-activated K+ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K+-selective (pCl/pK=0.046; PNa/PK=0.027). Channels incorporated into the bilayer with two orientations. In 65 % of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca2+ concentration of about 15 μmol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca2+ activated the channel. These results resemble those found for Ca2+-activated K+ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.

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Ca2+-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers

C a 2 + - a c t i v a t e d K + c h a n n e l s f r o m an insulin-secreting cell line i n c o r p o r a t e d i n t o planar lipid bilayers Y. Oosawa 0 S. J. H. Ashcroft 0 F.M. Ashcrofl 0 0 University Laboratoryof Physiology and 2Nuffield Department of ClinicalBiochemistry, University of Oxford , UK Summary. This study evaluates the use of the planar lipid bit layer as a functional assay of Ca2*-activated K § channel activity for use in purification of the channel protein. Ca2+-activated K* channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/1 KC1 and the channel was strongly K § (Pcl/PK----0.046; PNa/P~= 0.027). Channels incorporated into the bilayer with two orientations. In 65 % of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca2§ concentration of about 15 gmol/1. Charybdotoxin greatly reduced the probability of the channel being open when added tO the side opposite to that at which Ca2§ activated the channel. These results resemble those found for Ca 2§ K + channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer. Ca-'+-activated K-channel; pancreatic Beta cell; H I T T15 cell; insulin-secreting cell line; planar lipid bilayer - 9 Springer-Verlag1992 Ca2§ K* channels have been found in all Beta cells a n d Beta-cell lines investigated [ 1 ]. T h e i r precise functional role remains somewhat controversial, although it is now generally agreed that they probably contribute to repolarization of the Beta-cell action potential [ 2, 3 ]. Evidence also suggests that they have little influence on the glucose-dependent oscillations in Beta-cell m e m b r a n e potential (slow w a v e s ) f r o m which the action potentials arise [4] and are not essential for glucose-induced electrical activity in Beta cells [ 5 ]. T h e probability of Ca 2§ K § channels being o p e n is increased both by intracellular Ca z+ and by depolarisation. T h e precise mechanism by which Ca 2§ and voltage interact to increase channel activity requires knowledge of the channel structure, which is not yet available. Although a n u m b e r of voltage-gated K-channels have been cloned using oligonucleotide probes based on the sequence of the Shaker K-channel, this approach has proved unsuccessful for the Ca 2+-activated K + channel [ 6 ], presumably due to lack of homology. In addition, expression cloning of the channel in oocyctes is limited by low expession [ 7 ]. A n alternative strategy is protein purification followed by N-terminal sequencing and construction of oligonucleotide probes for screening of c D N A libraries. F o r this approach, it would be useful to have a functional assay which could be used to detect the presence of the Ca 2+-activated K § channel during protein isolation. Reconstitution of solubilised channel proteins in lipid vesicles followed by fusion with planar bilayers and single channel recording is one such m e t h o d [ 8 ]. In this paper we assess the usefulness of this technique as a functional assay for the Beta-cell Ca 2§ K § channel. As a source of tissue we have used the clonal Beta-cell line H I T T15 (HIT). This cell line was derived by SV40 transformation of hamster islet cells [ 9 ] and has many of the properties of normal Beta cells; in particular, secretion of insulin in response to glucose [ 9-11 ]. Patch clamp studies have established that H I T cells possess Ca z§ vated K § channels [ 12 ]. Materials and methods Cell culture HIT T15 cells (passage numbers 75-90) were cultured, passaged and harvested as described by Ashcroft et al. [ 10 ]. Membrane preparation Membranes were prepared from HIT T-15Beta cellsas described by Gaines et al, [ 13 ], with slight modification. Briefly, HIT cells were collected from confluent flasks and washed twice with phosphate1.0 1.0 1.0 buffered saline (Life Technologies, Paisley, UK). Cells were resuspended in ice-cold 5 retool/1Tris base (pH 8.0) containing a cocktail of protease inhibitors (0.1 retool/1 phenylmethylsulfonyl fluoride, 1 mmol/1EDTA, 1 mmol/1 iodoacetamide, 10 gg/ml soybean trypsin inhibitor and 10 gmol/1 leupeptin) and incubated on ice in a glass homogenizer for 40 min. The cells were then homogenized and the homogenate was centrifuged for 10 rain at 900 g. The supernatant was collected and centrifuged at 96000 g for 30 rain at 4~ The pellets were resuspended at a concentration of 1-2 mg/ml protein in 0.4 mol/1 sucrose, 10 mmol/1 Hepes-KOH (pH 7.1), frozen immediately in liquid nitrogen and stored at - 70~ Planar lipid biIayers Planar lipid bilayers were formed from a mixture of 50 % phosphatidylethanolamine (bovine heart) and 50% phosphatidylserine (bovine brain) dissolved in decane at a concentration of 25 mg/ml. All lipids were obtained from Avanti Polar Lipids (Birmingham, Ala., USA). Planar lipid bilayers were formed by painting the phosph01ipid solution across a 300-400 pm diameter hole in a polystyrene partition separating two solution-filled chambers of 5 ml (cis) and 3 ml (trans)and allowed to thin spontaneously [ 14,15 ]. The cis chamber (to which the vesicles were added) was voltage clamped at various potentials relative to the trans chamber: the trans chamber was grounded. All potentials refer to the potential of the cis chamber with respect to the trans chamber (i. e. cis-trans voltage). Vesicle fusion was deemed to have occurred in those bilayers in which channel activity was observed. In this paper, we only discuss those bilayers in which vesicle fusion occurred (n = 151). Data analysis Single channel currents were recorded under voltage clamp using a standard current-to-voltage amplifier and recorded on FM tape or video tape for !ater analysis. The frequency response of the system was 200 Hz. The currents were later amplified ( 10 or 50) and filtered at 200 Hz using an 8-pole Bessel filter (Frequency Devices, Springfield, Mass., USA). They were then digitised at 1 kHz using an Axolab analogue-to-digital converter and analysed using an IBM AT computer and the program PCLAME The probability of the channel being open (the open probability) was determined from amplitude histograms constructed from all data points. The records have been redisplayed using a Gould 3200 chart recorder. Solutions Both the cis and trans solution initially contained 140 mmol/1 KC1 and 10 mmol/1HEPES (titrated to pH 7.1 with KOH: additional K § about 2.5 mmol/1). To the cis chamber 1 mmol/1 CaC12 was added, followed by the vesicles (final protein concentration 2-10 gg/ml). Free calcium concentrations were adjusted by the subsequent addition of E G T A (titrated to pH 7.4 with K O H ) and the pH was subsequently readjusted to 7.1 with K O H (Table 1). Free Ca2§ concentrations were calculated using the binding constants of Martetl and Smith [ 16 ]. In some experiments NaC1 was partially substituted for KC1, as described in the text. Experiments were done at room temperature of 20-25 ~ Statistical analysis Results are given as mean _+the standard error of the mean (SEM). Results C a ; + - a c t i v a t e d K + c h a n n e l s w e r e s e e n i n 73 o u t o f t h e 151 b i l a y e r s i n w h i c h f u s i o n o c c u r r e d . T h e c h a n n e l w a s a b l e to i n s e r t i n t o t h e b i l a y e r w i t h its i n t r a c e l l u l a r s i d e f a c i n g e i t h e r t h e cis side o r t h e t r a n s s i d e o f t h e b i l a y e r . T h e i n c i d e n c e o f t h e C a 2+( c i s ) - a c t i v a t e d K § c h a n n e l w a s 64.6 % a n d t h a t o f t h e C a Z + ( t r a n s ) - a c t i v a t e d K § c h a n n e l w a s 35.4 % . W e f o c u s o u r a n a l y s i s p r i n c i p a l l y o n t h e p r o p e r t i e s o f t h e C a 2+ ( c i s ) - a c t i v a t e d K § c h a n n e l , w h i c h w a s d i s t i n g u i s h e d b y t h e p r o f o u n d d e c r e a s e i n its a c t i v i t y p r o d u c e d b y a d d i n g E G T A t o t h e cis s i d e o f t h e m e m b r a n e . Permeability F i g u r e 1 A s h o w s s i n g l e c h a n n e l c u r r e n t s r e c o r d e d at diff e r e n t m e m b r a n e p o t e n t i a l s in s y m m e t r i c a l 140 m m o l / 1 KC1 s o l u t i o n s at a cis C a 2+ c o n c e n t r a t i o n o f i mmol/Io c -- i~][[~ml]~Jl~l~]l~l~]m -20mVo-- ~ l l l ~ l ~ ' ~ l ~ t ~ M -40mVc-- ~ o-lOpA [ _ _ ls B -40 / I -20 / 2~0 v (mY) , ! .!~.=~. -20mY C-o_ !W ll lH_ im -40mY A (3olOpA i mmol/l 15.6 ~mol/l 0.25 ~mol/l CO O4 4 4 I I I I I 1 I I -80 -60 -40 -20 0 20 40 60 V (mY) B "~ L0 The m e a n single channel current-voltage ( I - V ) relation m e a s u r e d under these conditions (Fig. 1 B) is linear, with a slope conductance of 233 picoSiemens (pS) and a reversal potential close to 0 mV. T h e m e a n single channel I - V relation m e a s u r e d with 250 mmol/1KC1 cis and 50 mmol/1 KC1 trans is also showfi in Figure 1B. The reversal potential of - 3 4 . 5 m V was close to the calculated K+-equilibrium potential ( - 4 0 m V ) and indicates that the channel strongly selects K + o v e r C I - : the permeability ratio, Pa/PK, was 0.046. U n d e r quasi-physiological ionic conditions (cis: 140 mmol/1 KC1 and trans: 135 mmol/1 NaCI, 5 mmol/1 KC1), the slope conductance b e t w e e n - 2 0 m V and + 20 m V was reduced to 149 pS (Fig. 1 B). However, the single-channel I - V relation is no longer linear and was b e t t e r fit by the G o l d m a n - H o d g k i n - K a t z equation: where F is the Faraday, R is the gas constant and T is the absolute t e m p e r a t u r e , V is the m e m b r a n e potential and I is the current. PK and PN, are the permeability to K + and N a +, respectively. [K]t.... and [K]c~ are the trans and cis K + concentrations, respectively and [Na]~.... and [Na]c~s are the trans and cis Na + concentrations, respectively. T h e , continuous line drawn through the open circles in Fig: ure 1B was fit to equation (1) using a least squares method. T h e best fit was o b t a i n e d with a K permeability of 3.42 x 10-'3 cm3/s and a PNa/PKratio of 0.027. This permeability ratio indicates that the channel is considerably less p e r m e a b l e to Na + than K +. In symmetrical 140 mmol/1 KC1 solutions the K permeability was 4.25 x 10- ~3cm3/s and with 250 mmol/1 KC1 cis and 50 mmol/1 KC1 trans, the K permeability was 3.66 x 10- 13 cm3/s. Effects of calcium and voltage Channel activity was d e p e n d e n t on both Ca 2+ and voltage. The effect of m e m b r a n e potential on single channel currents and the probability of the channel being o p e n (open probability) is shown in Figure 2. Making the cis potential m o r e positive increased the channel o p e n probability: at a cis Ca 2+ concentration of 16 gmol/1, s o m e channel activity was evident at - 4 0 m V and the o p e n probability was half maximal at a b o u t , + 20 inV. As the cis Ca 2+ concentration was raised, the relationship b e t w e e n the o p e n probability of the channel and m e m b r a n e potential shifted to m o r e negative m e m b r a n e potentials, so that at any given potential channel activity was greater. The opposite e f f e c t was found w h e n the cis C a 2+ concentration was lowered and no channel activity was observed with 0.25 gmol/1 Ca 2+even at + 40 inV. C o n t r o l 12 n m o l / l C T X 0-O-O-C-lOpAI ~ 5s 40mY C h a r y b d o t o x i n ( C T X ) is a p o t e n t b l o c k e r o f high cond u c t a n c e Ca z+-activated K + channels [ 5, 17, 18 ]. Figure 4 shows that 12 nmol/1 C T X greatly r e d u c e d the c h a n n e l o p e n probability w h e n a d d e d to the side o p p o s i t e to that at which C a 2+ activated the channel. C T X i n d u c e d a longlived closed state, which separates the c h a n n e l openings into bursts. Ca2+(trans)-activated K +channel A s discussed, the C a 2+ - a c t i v a t e d K + c h a n n e l was also able to i n c o r p o r a t e into the bilayer with its intracellular side facing the trans side o f the bilayer. In this case, c h a n n e l activity was n o t influenced by the addition of E G T A to the cis side but was sensitive to variation o f the trans Ca 2+ c o n c e n t r a t i o n . Figure 5 A shows single c h a n n e l currents r e c o r d e d at different m e m b r a n e potentials in s y m m e t r i c a l 140 mmol/1 KC1 solutions in the p r e s e n c e o f a trans free calcium conc e n t r a t i o n o f 1 mmol/l. T h e m e a n I - V relation, given in Figure 5 B, reverses at 0 m V a n d has a slope c o n d u c t a n c e of 269 pS. T h e m e a n reversal potential shifted to - 36.0 m V w h e n the cis solution c o n t a i n e d 250 mmol/1KC1,1 retool/1CaC12 a n d the trans solution c o n t a i n e d 50 mmol/1 KC1 (Fig. 5 B) giving a p e r m e a b i l i t y ratio, Pa/PK, of 0.031. T h e m e a n single c h a n n e l c o n d u c t a n c e with these solutions was 225 pS. T h e Ca 2+ sensitivity o f the c h a n n e l r e s e m b l e d that o f the CaZ+(cis)-activated K + channel. Likewise, positive potentials applied t o the Ca 2+-activated side o f the bilayer increased the c h a n n e l o p e n probability (i. e., cis h y p e r p o larization). T o g e t h e r with the similarity of the single channel c o n d u c t a n c e and the K +-selectivity, these results confirm o u r idea that this c h a n n e l is the Ca > (cis)-activated K § c h a n n e l with a different o r i e n t a t i o n in the bilayer. Discussion Ca 2+-activated K § channels h a v e already b e e n r e p o r t e d in p a t c h clamp e x p e r i m e n t s o n the insulin-secreting cell lines H I T T15 [ 12 ] and R I N m 5 F [ 19 ] and in m o u s e [ 3 ] and rat p a n c r e a t i c B e t a cells [ 20-22 ]. T h e single c h a n n e l cond u c t a n c e of - 2 3 0 pS f o u n d in o u r studies falls within the range o f 211-250 pS r e p o r t e d in these previous studies ( m e a s u r e d in s y m m e t r i c a l 1 4 0 - 1 5 0 m m o l / 1 KC1 solutions). T h e r e p o r t e d CaZ+-sensitivity o f Ca2+-activated K + channels in B e t a cells, m e a s u r e d in p a t c h clamp experiments, varies b o t h b e t w e e n species and also f r o m p a t c h to patch. W h e n m e a s u r e d in inside-out patches excised f r o m H I T cell m e m b r a n e s , c h a n n e l activity at 0 m V was half-maximal at a b o u t 1 gmol/l Ca 2+ [ 12 ]. In o u r o b s e r v a tions the calcium sensitivity was s o m e w h a t lower: K0.5 = - 1 6 gmol/1 at 20 mV. O n e possible r e a s o n m a y be that o u r solutions did n o t contain M g 2+, w h e r e a s in the p a t c h clamp experiments, the intracellular solution contained 1.1 mmol/1 M g 2§ [ 12 ]. M g 2+ is k n o w n to increase the Ca 2§ o f Ca 2+-activated K * channels [ 23 ]. T h e C a 2+-sensitivity we m e a s u r e is still g r e a t e r than that f o u n d in m o u s e B e t a cells [ 20-22 ], however, which m a y explain w h y the Ca z+-activated K ~ current contributes a larger fraction o f the d e l a y e d o u t w a r d c u r r e n t in H I T cells [21. I n summary, we have s h o w n that the p r o p e r t i e s of Ca2+-activated K + channels f r o m H I T cell m e m b r a n e s , i n c o r p o r a t e d into p l a n a r lipid bilayers, do not differ significantly f r o m those r e p o r t e d for these channels in native m e m b r a n e s . This suggests that reconstitution o f Ca 2+-activated K + channels in p l a n a r lipid bilayers m a y be used as a functional assay to follow the c h a n n e l during p r o t e i n purification. Acknowledgements. We thank the Medical Research Council, the British Diabetic Association, the E. E Abraham Trust and the Royal Societyfor support. E M. A. was a Royal Society 1983University Research Fellow. The work described here has been carried out as part of a collaborative research programme with Drs. C. Betsholtz and M. Welsh (Uppsala University, Sweden), P.Rorsman (Gothenburg University, Sweden) and R-O.Berggren (Karolinska Institute, Sweden). We thank Dr. C. Miller of Brandeis University (Boston, Mass., USA) for his generous gift of charybdotoxin. We thank Drs. P.A. Smith, I. Niki, K. Furuya and C. Fewtrell for helpful comments. 1. Asheroft FM , Rorsman P ( 1990 ) Electrophysiology of the pancreatic ~-cell . Prog Biophys Molec Bio154 : 87 - 143 2. Satin LS , Hopkins WF , Fatherazi S , Cook DL ( 1989 ) Expression of a rapid, low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering . J Membr Bio1112 : 213 - 222 3. Smith PA , Bokvist K , Arkhammar P , Berggren PO , Rorsman P ( 1990 ) Delayed rectifying and calcium-activated K + channels and their significance for action potential rep01arization in mouse pancreatic ~cells . J Gen Physio195 : 1041 - 1059 4. Henquin JC ( 1990 ) Role of voltage- and Ca > -dependent K § channels in the control of glucose-induced electrical activity in pancreatic B-cells . Pfltigers Arch 416 : 568 - 572 5. 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Martell AE , Smith RM ( 1974 ) Critical stability constants , vol. 1 , Amino acids , vol. 2 , Amines . Plenum Press, New York 17. Miller C , Moczydlowski E , Latorre R , Phillips M ( 1985 ) Charybdotoxin, a protein inhibitor of single Ca>-activated K § channels from mammalian skeletal muscle . Nature 313 : 316 - 318 18. Miller C ( 1988 ) Competition for block of a Ca2+-activated K § channel by charybdotoxin and tetraethylammonium . Neuron 1 : 1003 - 1006 19. Velasco JM , Petersen OH ( 1987 ) Voltage-activation of high-conductance K +channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration . Biochim Biophys Acta 896 : 305 - 310 20. Cook DL , Ikeuchi M , Fujimoto WY ( 1984 ) Lowering ofpH~ inhibits Ca2*-activated K + channels in pancreatic B-cells . Nature 311 : 269 - 271 21. Findlay I , Dunne MJ , Petersen OH ( 1985 ) High-conductance K + channel in pancreatic islet cells can be activated and inactivated by internal calcium . J Membr Bio183 : 169 - 175 22. Tabcharani JA , Misler S ( 1989 ) Caz+ -activated K ~channel in rat pancreatic islet B cells: permeation, gating and blockade by cations . Biochim Biophys Acta 982 : 62 - 72 23. Golowasch J , Kirkwood A , Miller C ( 1986 ) Allosteric effects of Mg2~ on the gating of Ca~+-activated K +channels from mammalian skeletal muscle . J Exp Bio1124 : 5 - 13 Received: 9 December 1991 and in revised form: 19 March 1992


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Y. Oosawa, S. J. H. Ashcroft, F. M. Ashcroft. Ca2+-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers, Diabetologia, 1992, 619-623, DOI: 10.1007/BF00400252