Electrophysiological modifications induced by the fluorescent probe, pyrene, on Myxicola giant axons

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Biochimica et Biophysica Acta, 597 (1980) 622--625 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 71447

ELECTROI'HYSIOLOGICAL MODIFICATIONS INDUCED BY THE F L U O R E S C E N T PROBE, PYRENE, ON M YXICOLA GIANT AXONS

H. DUCLOHIER, J.P. DESMAZES and D. GEORGESCAULD

Centre de Recherches Paul Pascal (CNRS), Domaine Universitaire, 33405 Talence (France) (Received October 22nd, 1979)

Key words: Pyrene; Conductance; Membrane capacity; (Giant axon)

Summary Current- and voltage-clamp experiments on Myxicola giant axons labelled with pyrene showed decreased Na ÷ and K ÷ conductances. The lowfrequency membrane capacity and the gating charge transfer were slightly reduced. It may be inferred that pyrene is incorporated in some hydrophobic membrane domains close to the ionic channels

It has been postulated that membrane fluidity might play a role in nerve excitation [1,2]. Now, the fluorescent hydrophobic probe, pyrene, has proved to be a useful tool for monitoring membrane fluidity [3,4] and has allowed us to show, on stimulated and labelled nerve fibres, transient changes of pyrene m o n o m e r and excimer emissions [5]. Unlike pyrene, the fluorescent probes already used by others workers [6] to obtain fluorescent signals during nerve excitation are not directly relevant for the membrane fluidity changes. They were also found to be non-toxic and potential-dependent. Before the investigation of pyrene fluorescence signals during voltage-clamp experiments, it was important to carry out an electrophysiological analysis to decide if the probe induced some functional perturbations. Giant axons from the annelid sea-worm, Myxicola infundibulum, were dissected and current- or voltage-clamped [ 7 ]. Modifications included an adjustable compensation for the series resistances. Gating charge transfer was measured from the asymmetry current yielded by the single-sweep procedure [8]. A separate set of impedance experiments, at resting membrane potential, has also been performed according to the method described for the squid giant axon [9]. As the effects of pyrene were particularly obvious with extensive labelling and since the latter required a long incubation (up to 2 h) with slight agitation at room temperature, the behaviour

623 of these labelled axons was compared to that displayed by a set of 'control axons' subjected to the same treatment, but in the normal physiological medium i.e., artificial sea-water. It must be stressed that these conditions were the standard procedure which had to be used for recording nerve extrinsic fluorescence either in the steady-state or during excitation. At each stage, the good condition of the fibres was tested by recording external diphasic action potentials. Labelling procedure using bovine serum albuminpyrene complexes and fluorescence measurements were performed as already described for crab leg nerves [10]. The extent of labelling was assessed by the pyrene m o n o m e r to excimer emissions ratio (Ie/Im) yielded from the fluorescence spectra of those labelled axons which had been subjected to the electrophysiological analysis. Fig. 1 summarizes the results obtained under current-clamp conditions. The membrane potential responses to short depolarizing shocks are shown (Fig. l a , b, c). The two upper oscilloscope records compare typical examples of action potentials for the most labelled axons (b) with the control (a). Note the reduced and wider spike, the increased threshold and the negative post-potential. In curve c, it is shown that the spike amplitude attained a m a x i m u m reduction (approx. 20%) as soon as Ie/Im a m o u n t e d to 0.15. On average, there was little or no alteration in resting membrane potential. The incubation of the axons with bovine serum albumin alone, at a concentration in artificial sea-water corresponding to the highest value used for the labelling solution (0.60 mg/ml), did not induce significant effects. The lower part of the figure (Fig. l d ) shows the membrane capacity, CM, plotted as a function of frequency, the range of which was limited to 20 kHz because of larger relative errors at higher frequencies. Data are corrected for the series resistances [9], being estimated as 20 ~ .cm 2 . On average, it appears that CM decreased by 10% or the frequency-dependent part by 20%. This is only obvious for low frequencies as both curves tend to superimpose above 10 kHz. Fig. 2a and b shows the voltage-clamped membrane currents at 0 mV, for a Myxicola giant axon in the control solution (a) and then for this same axon, 40 min after external exposure to 1 ml of the most concentrated solution of bovine serum albumin-pyrene in artificial sea-water (b). In both records, the temperature was first 8°C and then it was shifted to 18°C yielding the larger traces. The main effects of pyrene incorporation are clearly visible, i.e., reductions of Na ÷ and K ÷ conductances, especially marked at the higher temperature. The remaining conductances a m o u n t e d to 75 and 50%, respectively, for gNa+ and gK÷ at 18°C. The temperature coefficient (Q,0) for gNa*, the Na* conductance, fell from the control value of 1.5 in (a) to 1.25 in (b). This last figure is surprisingly close to Qlo Of Ie/Im (1.29 in the same temperature range) as measured from fluorescence experiments (unpublished results). Fig. 2 also indicates the lack of effect on the Na ÷ conductance kinetics. Indeed, neither the time-to-peak nor the time course of the early current decay (and consequently, neither Tm nor Th, the Na ÷ activation and inactivation time constants, respectively) were significantly affected. In order to see if the partial inhibition of the Na ÷ conductance was due to a decreased gating charge transfer, we attempted measurements of

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Fig. 1. M y x i c o l a g i a n t a x o n s u n d e r c u r r e n t - c l a m p c o n d i t i o n s . R o o m t e m p e r a t u r e 1 8 - - 2 0 ° C. A f t e r i n c u b a t i o n w i t h b o v i n e s e r u m a l b u m i n - p y r e n e c o m p l e x e s , t h e l a b e l l e d fibres w e r e w a s h e d t w i c e f o r 3 0 m i n in K +free artificial Sea-water. (a) R e s t i n g p o t e n t i a l level a n d s p a c e - c l a m p a c t i o n p o t e n t i a l f r o m a ' c o n t r o l ' a x o n ; t h e just s u b - t h r e s h o l d r e s p o n s e is also s h o w n . (b) T h e s a m e p a r a m e t e r s f o r a n e x t e n s i v e l y l a b e l l e d a x o n ( I e / I m = 0 . 2 2 ) . G r a p h c s u m m a r i z e s the m e a n b e h a v i o u r f o u n d as r e s u l t o f t h r e e increasing p y r e n e labellings. T h e a c t i o n p o t e n t i a l a m p l i t u d e (+) a n d the t h r e s h o l d (X) are s h o w n . G r a p h d is a p l o t of m e m b r a n e c a p a c i t y , CM, as a f u n c t i o n o f f r e q u e n c y . T h e p e a k - t o - p e a k a m p l i t u d e o f t h e s i n u s o i d a l oscillations o f m e m b r a n e p o t e n t i a l w a s s e t t l e d a t 4---6 inV. O n l y t h e f r e q u e n c y - d e p e n d e n t p a r t o f C M (i.e., a b o v e 0 . 5 / z F / c m 2 ) is s h o w n . D a t a are c o r r e c t e d f o r t h e series resistances, R S = 20 ~2°cm 2 , e s t i m a t e d f r o m t h e h i g h - f r e q u e n c y l i m i t o f resistance: ( e ) a v e r a g e d f r o m t h r e e r a w a x o n s as a c o n t r o l a n d (o) a v e r a g e d f r o m t h e s a m e n u m b e r of p y r e n e labelled a x o n s ( I e / I m a p p r o x . 0 . 2 0 ) . V e r t i c a l bars s h o w t h e s t a n d a r d e r r o r o f t h e m e a n . Fig. 2 . ( a , b ) M e m b r a n e c u r r e n t s f o r a d e p o l a r i z i n g pulse t o 0 m V ( M y x i c o l a g i a n t a x o n ) a t 8 a n d 1 8 ° C : (a) c o n t r o l in K+-free artificial Sea-water a n d (b) 4 0 m i n a f t e r e x p o s u r e to t h e m o s t c o n c e n t r a t e d b o v i n e s e r u m a l b u m i n - p y r e n e s o l u t i o n (5 o 10 - s M in artificial s e a - w a t e r ) . R e c o r d s c o r r e c t e d for l e a k w h i c h was una f f e c t e d . H o l d i n g p o t e n t i a l V H = - - 8 0 m V ; c o m p e n s a t i o n f o r R S ( 1 0 ~ o c m 2 ). ( c , d ) O f f - a s y m m e t r y c u r r e n t s , r e s p e c t i v e l y , for a c o n t r o l a n d a p y r e n e - l a b e l l e d a x o n , b o t h b a t h e d in K+-f~ee artificial Sea-water + 3/~M t e t r o d o t o x i n + 10 m M 4 - a m i n o - p y r i d i n e . H o l d i n g p o t e n t i a l VHffi--100 m V a n d a m p l i t u d e of v o l t a g e s t e p s = -+100 inV. T h e c h a r g e t r a n s f e r , Q o f f ( r e t u r n o f gating c h a r g e s t o t h e i r original posit i o n ) , has b e e n c a l c u l a t e d as t h e a r e a u n d e r t h e t r a c e e x t r a p o l a t e d t o z e r o t i m e ( e n d o f pulses): t e m p e r a t u r e 8°C.

asymmetry or displacement current by a single-sweep subtraction of currents yielded by a pair of opposite voltage pulses. Because the off-current trace was found to be less noisy and somewhat more stable than the oncurrent, we chose to calculate the charge transfer, Qoff, assuming for this pulse width (1 ms) that Qoff=Qon. Fig. 2c and d shows that the ratio, Qoff(labelled)/Qoff(control), was approx. 0.65. There is good agreement between this figure and the impedance results. As a matter of fact, if one is interested by the relative change of the capacity component, CM(LF_HF), responsible for the low-frequency (LF) dispersion and hypothetically specific

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of the proteinous channels, it was found (Fig. l d ) that CM(LF--HF)(labelled)/CM(LF_HF)(control) = 0.75--0.8. On the whole, there seems to be no d o u b t that pyrene exerts some irreversible constraints on membrane conductances and on the gating charges, possibly by immobilization of a fraction of them (up to 30%). These effects resemble those displayed by some local anaesthetics like procafne [11 ]. The lipid bilayer in which the 'voltage-sensors' of excitable channels have been assumed to protrude [2] seems to be the main candidate for pyrene inclusion, according to its solubility characteristics, although one cannot exclude a direct incorporation into some channels' hydrophobic sites. Further experiments are in progress to decide the ultimate mechanism (block of a fraction of the channels' population or reduction of single channel conductance) and to eventually correlate these electrophysiological effects with structural perturbations. It may already be inferred that pyrene incorporation does not significantly affect membrane thickness, since the highfrequency limit of membrane capacity appeared unchanged. These results differ from those obtained with squid giant axons and with solvent-free phosphatidylcholine-cholesterol treated with n-alkanes [12,13]. As regards to the search for specific fluorescence signals and the involvement of membrane fluidity in the excitation process, as still postulated recently [14,15], the above reported sensitivity of excitable channels towards pyrene may be considered as positive information although it stresses some limitations, particularly for the concentration range, in the use of the 'probe methods'. We thank Dr. H. Meves and Dr. J.I. Gillespie from the Plymouth Marine Biological Laboratory for helpful comments. The technical assistance of J. Piaud and J. Audin is acknowledged.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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