Calcium current reactivation after flash photolysis of

Transcription

1 Journal of Physiology (1995), 487.1, pp Calcium current reactivation after flash photolysis of nifedipine in skeletal muscle fibres of the frog D. Feldmeyer, P. Zollner, B. Pohl and W. Melzer * Ruhr- Universitdt Bochum, Lehrstuhl fiur Zellphysiologie, ND 4, Germany D Bochum, 1. L-type calcium currents were activated by depolarization of cut muscle fibres of the frog. The current was blocked by the dihydropyridine compound nifedipine (5-10,tM) and reactivated by flash photolysis of the drug. 2. In the presence of nifedipine, increasing the time interval between the onset of depolarization and the flash resulted in progressively faster kinetics of the flash-induced current. This change developed with a slow time course similar to that of normal current activation. 3. A fast gating mode of the normally slow L-type channel was induced by conditioning activation (500 ms prepulses) applied 80 ms before a test step to the same potential. After block by nifedipine, flash-photolysis was carried out 40 ms before the end of the long conditioning pulse. The flash-induced current had the same rapid time course as the current activated by the subsequent test voltage step. 4. Similarly, the time course of current activation was comparable for the voltage-induced fast mode activation (flash applied 5 ms before the test step) and the flash-induced activation 40 ms after the onset of the test depolarization. 5. Our data suggest that in frog skeletal muscle nifedipine inhibits calcium current activation by blocking a rapid channel gating step while the slow conformational change that normally limits the rate of activation of the L-type calcium channel remains unaffected. UV flash illumination results in a fast reactivation indicating that the channels need not be inactivated to be blocked by nifedipine. The dihydropyridine-sensitive Ca2P channel (DHP-receptor) of skeletal muscle is one of the most thoroughly characterized transmembrane proteins (Glossmann & Striessnig, 1990; Lamb, 1992). Depolarization results in the activation of a slow DHP-sensitive (L-type) Ca2P inward current. The slow activation kinetics has been attributed to properties of the first internal repeat of the al-subunit of the DHP receptor (Tanabe, Adams, Numa & Beam, 1991). In frog muscle fibres (Feldmeyer, Melzer, Pohl & Zollner, 1990b, 1992; Garcia, Avila-Sakar & Stefani, 1990) and reconstituted transverse tubular membranes of rabbit muscle (Ma, Hosey & Rios, 1992), conditioning depolarizations led to rapid voltage-dependent current activation. To explain this behaviour, models have been suggested which include slow and fast gating steps associated with the four homologous internal repeats of the al-subunit (Feldmeyer et al. 1992; Ma et al. 1992). A variety of drugs that bind to the al-subunit of the DHP receptor have been of importance for both isolation and functional characterization of the protein. Light-sensitive dihydropyridines, like nifedipine, are particularily useful for functional studies because they allow the rapid inactivation of the ligand by applying a strong UV light flash (Morad, Goldman & Trentham, 1983; Gurney, Nerbonne & Lester, 1985). In the present study we investigated how dihydropyridines interact with the proposed activation gating mechanisms by combining voltage-clamp measurements of the slow L-type Ca2+ current in frog muscle with rapid UV flash photolysis of the dihydropyridine channel antagonist nifedipine. * To whom correspondence should be addressed at Universitat Ulm, Abteilung fur Angewandte Physiologie, Albert-Einstein-Allee 11, D Ulm, Germany. This manuscript was accepted as a Short Paper for rapid publication.

2 52 D. Feldmeyer, P Zollner, B. Pohl and W Melzer J Phy8iol METHODS Voltage clamp of isolated cut muscle fibres Short segments of isolated twitch muscle fibres were dissected from leg muscles (m. semitendinosus or m. iliofibularis) of frogs (Rana temporaria and Rana esculenta) which were killed by cervical dislocation and decapitated. Fibres were mounted in a double Vaseline gap set-up for voltage clamping described previously (Feldmeyer, Melzer & Pohl, 1990a). The composition of the artificial internal solution was (mm): caesium glutamate, 50; tetraethylammonium (TEA) glutamate, 30; MgCl2, 6 2; Cs2-EGTA, 20; Na2-ATP, 5; glucose, 5.6; Cs-Hepes, 10 (ph 7 0). In addition the solution contained 0 4 mm of the metallochromic indicator Antipyrylazo III, the absorbance of which was measured to monitor internal perfusion. The external solution contained (mm): Ca(CH3SO3)2, 10; TEA(CH3SO3)2, 120; 4-aminopyridine, 1; anthracene-9-carbonic acid, 0 5; tetrodotoxin, 3-1 x 10-4; TEA- Hepes, 2 (ph 7 4). A 1 mm stock solution of nifedipine was prepared in dimethyl sulphoxide and diluted in the external solution to the final concentration. The membrane was held at a potential of -80 mv and depolarizing voltage pulses were applied. Pulse application and recording of membrane voltage and current were carried out using an LSI-11 computer system (SMS 1000; Scientific Micro Systems) equipped with 12 bit D/A and A/D boards (DT2766 and DT2782; Data Translation). Records were sampled at rates between 0-1 and 1 khz. In order to correct for linear resistive and capacitive current components negative control pulses of one-quarter of the test pulse amplitude were applied, and the corresponding current (usually the average of 8 sweeps) was multiplied by four and added to the test current. Flash photolysis of nifedipine For photochemical inactivation of nifedipine we applied brief intense UV light flashes to the voltage-clamped fibre segment. A xenon arc lamp system (JML; G. Rapp, Dossenheim, Germany) was used as the UV light source. The optical axis of the flash lamp system formed an angle of about 45 deg with the surface of the solution in the external pool of the experimental chamber. A broad UV light band (centred at 320 nm) was selected by a bandpass filter (UG1 1; Schott). Light pulses generated by the flash lamp were triggered during the data acquisition period within a specified sampling interval. The intense UV radiation decayed with a half-time of less than 1 ms (see Rapp & Giith, 1988). The flashes were separated by time intervals of several minutes thus allowing recovery of the block by diffusional exchange with the large pool of unilluminated nifedipine in the external compartment of the chamber. The figures show the light-dependent difference current, i.e. the difference between consecutively recorded currents before and after UV irradiation. A mv 2 na nf-1 B E 300 d e f _.- T DC.) ~0.I.. 0 _-I co 0 0) E _C I tflash (ms) tflash Figure 1. Reversal of nifedipine block by flash photolysis after different durations of depolarization A, calcium inward currents before (b) and after (c) block by 5juM nifedipine, and following flash photolysis (marked by arrows) 10 ms before (d) and at two different times during (e and f ) identical test depolarizations (a). The interval t1.h between the onset of the depolarization and the application of the light flash was 50 ms for e and 500 ms for f. B, half-time of current reactivation after relief of nifedipine block at different tf1*ch values; the value at time 0 is the average of one control measurement without nifedipine (320 ms) and two different flash applications 10 ms before the voltage step (269 and 308 ms). Linear capacitance, 29f6 nf; temperature, 17f 'C.

3 J Phy8iol Flash photolysis of nifedipine in skeletal muscle 53 RESULTS In the experiment shown in Fig. 1, voltage clamp depolarizations to -20 mv of 1 s duration were applied from a holding potential of -80 mv. The slow Ca2P inward current (L-type) that was activated by these pulses (Fig. la b) was blocked completely by 5/M nifedipine (Fig. la c) applied to the external pool of the chamber. When an intense UV light flash was applied immediately before the step depolarization a considerable fraction of the original current amplitude could be activated with the same slow time course (Fig. 1A d). Subsequently, nifedipine was photolysed at different times after the onset of a depolarization. It can be noticed that the time course of activation was slow when the flash was applied only 50 ms after the depolarization (Fig. IA e) but became considerably faster when the interval between voltage step and flash was increased to 500 ms (Fig. laf). The half-time of reaching the current maximum after a flash is plotted in Fig. 1B against different intervals, tflash, between the onset of the depolarization and the UV flash. The gradual decrease of the activation half-time with tflash reveals a slow reaction of the DHP receptor that is initiated by the depolarization but cannot be observed due to the drug-induced current block. It is apparent that the decline of the half-time of the flash-activated current displays a time course similar to the slow current activation in the absence of block. This type of experiment was carried out in nine skeletal muscle fibres (3 with 5 um and 6 with 10 /SM nifedipine). In all cases the activation half-time was considerably reduced with increasing tflash' on average from to 10A ms (mean+ S.D.; i.e. to 5% of the control value; pulse voltage: -20 mv). A possible explanation for this behaviour would be a block of the pore which had no effect on the voltage-dependent gating of the channel. Flash photolysis of nifedipine in the drug-bound conformational state of the channel equivalent to the open conformation should then lead to current flow with a time course determined by the photochemical destruction of nifedipine and the dissociation of the photoproducts. However, while photolysis of nifedipine has been reported to occur in less than 100,us (Morad et al. 1983), the lower limit for the flash-induced current activation at long depolarizations was about 100 times larger (-10 ms; see also Fig. 3). Thus, the channel appears not to open immediately after nifedipine photolysis. This suggests that before opening the channel has to undergo a structural change which is much faster than the normal voltage-dependent activation but still considerably slower than the photolysis reaction. Previously, we have presented evidence for a rapid, voltagedependent step in the gating of the skeletal muscle L-type channel which has considerably faster kinetics than the slow A a -80 mv b 0mV 500ms B +20 mv a 500 ms -80 mv -60 mv 80 ms 5 na nf-1 b c o mv 1 na nf-1 d -60 mv 80 ms c A,- Figure 2. Fast gating of the L-type channel and flash-induced current recovery at the same voltage A, a fast gating mode (b) is evoked by a prepulse protocol (a) which includes a 500 ms conditioning depolarization (O mv), an 80 ms repolarization interval (-60 mv) and a test depolarization to 0 mv (Feldmeyer et al. 1990b). The time interval marked with the bar is shown at higher time resolution in c and d. Linear capacitance, 18-7 nf; temperature, '0 'C. B, double-pulse sequence (a) similar to the one in A applied to a nifedipine-blocked fibre. Flash photolysis (marked by arrows) either precedes the test depolarization by 5 ms (b) or follows the onset of the test depolarization by 40 ms (c). In both cases the current activation shows a similar time course. The rapid spikes are artifacts caused by the flash lamp. Linear capacitance, I1 2 nf; temperature, 17'2 'C.

4 54 D. Feldmeyer, P Zollner, B. Pohl and W Melzer J Phy8iol time course of current activation (Feldmeyer et al. 1990b, 1992). Shortly after a strongly depolarizing prepulse the current exhibits rapid gating (Feldmeyer et at. 1990b; Garcia et al. 1990). The purpose of the experiments shown in Figs 2 and 3 was to compare the time course of this fast voltage-dependent gating with that of the fastest flashinduced activation at the same potential. The voltage-clamp protocol used to induce the fast gating mode of the channel (Feldmeyer et al. 1990b, 1992) is shown in Fig. 2A a. A strong 500 ms long depolarization to 0 mv was followed by a partial repolarization (for 80 ms) to -60 mv, a subthreshold potential that leads to complete current deactivation. However, current activation during the subsequent depolarization is much more rapid than during the first pulse. Figure 2A c and d shows the repolarization interval (indicated by the bar in Fig. 2A a) in a second measurement with higher time resolution. After block of the current by nifedipine, UV light flashes were applied at specific times during the pulse protocol. In Fig. 2B b the flash was applied during the repolarization interval, i.e. 5 ms prior to the second depolarization. Although the current was blocked by nifedipine during the prepulse the current activation by the second depolarization was fast (see also Feldmeyer et at. 1992). In Fig. 2B c the flash was applied 40 ms after the onset of the second depolarization, i.e. at a time when the voltage-dependent activation of the unblocked current in Fig. 2B b had almost reached its maximum. Unlike the experiment shown in Fig. 1A, the activation of the flash-induced current was not much faster than the voltage-induced current in Fig. 2B b. Single exponential functions were fitted to the activation time courses within an interval of 36 ms starting 4 ms after the trigger of the flash or the onset of the test pulse. In three experiments of this kind we obtained time constants of and ms for the depolarizationinduced and flash-induced current, respectively. Figure 3 illustrates one of three experiments in which the flash was applied 40 ms before the end of the conditioning depolarization, i.e. at a time when the unblocked current was maximally activated. Thus, the flash-induced and the following depolarization-induced activation were separated by only 120 ms. The rate of activation of the flash-induced current (interval 1; Fig. 3B) was very similar to that of the current during the subsequent depolarization (interval 2); this is illustrated in Fig. 3C in which both currents are superimposed by shifting interval 1 (bold line) to the right such that the moment of triggering the flash and the start of the depolarization coincide (arrow). Single exponential fits to the currents gave activation time constants of (flash) and ms (voltage step). DISCUSSION The results obtained in this study give some insight into the interaction of dihydropyridines with L-type Ca2P channel gating in frog skeletal muscle. We have demonstrated that the speed of flash-induced current activation is dependent on the period of time the membrane was depolarized prior to the application of the flash. If nifedipine binds only to the inactive state of the dihydropyridine receptor (see for instance the model proposed by Bean, 1984, for cardiac L-type Ca2+ channels) and therefore blocks the channels at a negative holding A B -80 mv +20mV 500 ms -60 mv 80 ms B 1 2 C 2- I 1 na nf-' 1 na nf'1 40 ms Figure 3. Comparison of flash-induced current recovery after a long preset depolarization and voltage-dependent activation in the fast gating mode A, pulse protocol (identical to the one in Fig. 2B) to induce fast mode of gating. B, flash-photolysis (marked by arrow) precedes repolarization from the 500 ms conditioning pulse by 40 ms. C, the flashinduced current (section labelled 1 in B) is superimposed (bold trace) on the voltage-induced current of the same record (section labelled 2 in B; thin trace) by shifting it 120 ms to the right. This shows the almost identical time course of activation. Same fibre as in Fig. 2B.

5 J Physiol Flash photolysis of nifediipine in skeletal muscle 55 potential by causing a shift into the inactive state, a flashinduced activation (as observed in our experiments) would not occur. At the membrane potentials used here and within the short observation intervals, no repriming of inactivated channels is to be expected. Block of the skeletal muscle L-type channel by selective binding to the inactive state was also ruled out by Neuhaus, Rosenthal & Liittgau (1990) because they did not observe the expected shift to more negative potentials of the steady-state inactivation curve. On the other hand, if nifedipine blocks the channels by selectively stabilizing the resting state and preventing the rate-limiting slow gating step, one would anticipate the same slow flash-induced activation independent of the duration of a preceding depolarization. Experiments similar to those shown in Fig. 1 have been performed on cardiac muscle cells which possess a more rapidly activating L-type current (Gurney et al. 1985). These results indicated that at the normal resting potential nifedipine binds preferentially to the resting state and blocks the current by stabilizing this state. No change in the activation kinetics of the Ca2+ current, as observed here, was reported. Instead, the rate of light-induced reactivation of the Ca2+ current was equal to the normal speed of activation. This is clearly different from our results where the rates change drastically (Fig. 1). The change in the Ca2+ current time course observed here indicates that during step depolarization, the nifedipine-bound channel molecule is still capable of undergoing a slow conformational change comparable to the normal slow gating transition, even though the channel does not conduct calcium. It is possible that the slow transition leads to a conformation similar to the open state of the channel (but blocked by the drug). If the relief of channel block occurs simultaneously with flash photolysis one would expect a very rapid opening (in the submillisecond range according to Morad et al. 1983) of those channels which have entered the drug-bound active state. This should lead to a very fast initial step in current. However, the fastest reaction observed was in the 10 ms range. This might be due to a more complicated photolysis scheme. For instance, channel-bound drug molecules might be protected from the light and would have to dissociate from the binding site first to allow ionic transport through the pore. The dissociation of drug molecules that are not photolysed would then be the ratelimiting step in channel opening. However, the striking similarity between the time course of current activation after photolysis and after a voltage step when the channel is in the fast gating mode induced by predepolarization (Figs 2 and 3) makes another explanation more likely. Nifedipine may prevent a rapid voltage-dependent transition which is necessary for channel opening (Fig. 4). This transition starts as soon as the drug is destroyed by UV irradiation. Under normal conditions, a second much slower reaction limits the gating speed. However, after a conditioning depolarization of sufficient duration (and strength) this slow reaction has already taken place and thus the fast reaction becomes rate-limiting when the flash is applied. The results of Gurney et al. (1985) obtained in cardiac muscle may differ from ours simply because nifedipine-sensitive and -insensitive gating reactions might have similar (rapid) kinetics in heart muscle. Binding sites of 1,4-dihydropyridines have been found in domains III and IV of the al -subunit (Regulla, Schneider, Nastainczyk, Meyer & Hofmann, 1991; Nakayama, Taki, Striessnig, Glossmann, Catterall & Kanaoka, 1991; Striessnig, Murphy & Catterall, 1991). Catterall & Striessnig (1992) suggest that DHPs act at the interface between repeats III and IV. These two domains are thought to participate in a rapid gating step (see models by Feldmeyer et al and Garcia, Tanabe & Beam, 1994) because a chimeric construct of the cardiac al -subunit containing repeats III and IV of skeletal muscle shows rapid voltage-dependent activation (Tanabe et al. 1991). Possibly binding of DHPs to these domains prevents a rapid step of voltage-dependent gating (Fig. 4; this does not rule out the presence of additional rapid gates which are unaffected by nifedipine). The fast reaction is normally Nifedipine OFF OFF Fast Slow ON ON OPEN Figure 4. Possible explanation of the results with a simple reaction scheme Gating of the DHP-sensitive calcium channel is assumed to involve at least two kinetically distinct reactions which are both necessary for channel opening. Binding of the DHP (to domains III and IV; Catterall & Striessnig, 1992) selectively prevents a fast gating process that normally precedes the slow, rate-limiting step of current activation. After a sufficiently long depolarization the slow step has reached its activated state (ON) and flash photolysis of nifedipine produces activation of the current with a rate which is now limited by the fast gating reaction.

6 56 D. Feldmeyer, P Zollner, B. Pohl and V4 Melzer J Physiol accompanied by a much slower step which limits the rate of channel opening and appears to involve domain I (Tanabe et al. 1991; Nakai, Adams, Imoto & Beam, 1994). Our results (Fig. 1) indicate that the slow gating reaction proceeds during depolarization in the DHP-blocked state. Upon flash photolysis the rapid step follows and leads to faster than normal current activation. According to this scheme, the state obtained immediately after flash photolysis is identical to the closed state reached initially after repolarization from a conditioning depolarization: the slow gate is activated (ON) but the fast gate is still deactivated (OFF) (Fig. 4 and Feldmeyer et al. 1992). Consequently, the time course of current activation during a new depolarization to the same potential is very similar (Figs 2 and 3) to the flash induced current reactivation. The fact that activation was slightly slower at the voltage step may be due to the time required to charge the membrane. In addition, a small fraction of channels may have returned from the conditioned state to the resting state within the 80 ms interval at -60 mv. In summary, we conclude that the slow transition which determines the time course of opening of the L-type calcium channels in skeletal muscle under normal conditions can proceed largely unaffected during nifedipine block while a second relatively fast channel transition necessary for channel opening is prevented. BEAN, B. P. (1984). Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proceedings of the National Academy of Sciences of the USA 81, CATTERALL, W. A. & STRIESSNIG, J. (1992). Receptor sites for Ca2 channel antagonists. Trends in Pharmacological Sciences 13, FELDMEYER, D., MELZER, W. & POHL, B. (1990a). 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Proceedings of the National Academy of Sciences of the USA 88, TANABE, T., ADAMS, B. A., NUMA, S. & BEAM, K. G. (1991). Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics. Nature 352, Acknowledgements We thank Dr H. Ch. Liittgau for his support and encouraging discussions. The work was supported by grants from the Deutsche Forschungsgemeinschaft (FG Konzell). Author's present address D. Feldmeyer: Max-Planck-Institut fur medizinische Forschung, Abteilung fur Zellphysiologie, JahnstraB3e 29, D Heidelberg, Germany. Received 16 May 1995; accepted 26 June 1995.