Abstract This paper examines kinetic properties of Na- channels in Strombus luhanus, a gastropod notable for its rapid backward jumping escape response. Previous comparative studies on cephalopod and gastropod Na- channels indicate that taxonomic differences exist in the voltage-dependencies of activation and inactivation: cephalopod channels are fast across a broad range of voltages, whereas gastropod channels are much slower at negative voltages than they are at very positive voltages. The voltage-independence of cephalopod Na+ channel kinetics is hypothesized to be important in mediating fast escape and swimming behavior. Analysis of kinetic parameters of Strombus sodium currents was performed in order to ascertain whether the voltage-dependence of their activation and inactivation rates deviate from those of slower gastropods and resemble those of cephalopods. It was found that as in cephalopods, activation rates in Strombus Na¬ channels display little voltage-dependence. Similar to other gastropods, however, inactivation rates in Strombus Na+ channels are quite voltage-dependent. Introduction The gastropod mollusk Strombus luhuanus exhibits a dramatic backward jumping escape response in the presence of molluscivorous cone snails. During an escape sequence, à Strombus thrusts its serrated operculum forward and into the substratum, lifts it shell off the ground, extends its muscular foot, and lands with its shell up to 5 centimeters backward from where it started (Berg 1974). Analysis of footage of the Strombus escape response reveals that a typical jump lasts approximately a second, from the moment the snail first lifts its shell from the substrate to the time it lands. Strombus' ability to rapidly initiate this escape sequence, combined with the considerable muscular control it exhibits during a single jump, suggests that the Strombus neuromuscular system may be capable of high-frequency activity. One means of examining the speed at which neurons can transmit information is to investigate the kinetic characteristics of voltage-gated ion channels responsible for the propagation of action potentials, particularly Na- channels. Kinetic properties of Strombus Na- channels are interesting not only in their own right, but also in comparison to those of other mollusks. Notably, cephalopod sodium channels possess fast activation and inactivation kinetics over a broad range of voltages, but kinetics of gastropod Na- channels, in contrast, are much more voltage-dependent (Gilly et al. 1997). Thus, gastropod Na¬ channels are slower at middle voltages (-20-0 mV) than they are at very positive voltages. It has been suggested that the fast Na- channel kinetics at mid-range voltages near action potential threshold, such as those in cephalopods, expedite rapid signaling crucial for control of fast swimming behavior or escape. Given Strombus' rapid escape response, it is interesting to ask if they possess "slow" or "fast" Nat channels at the mid range of voltages. This paper describes the results of experiments investigating both Strombus' ability to transmit high frequency motor information as well kinetic parameters of sodium channels recorded from Strombus pedal ganglia neurons. This paper integrates data on Strombus sodium channels with similar data on Loligo and Aplysia taken from Gilly et al. (1997). While Strombus sodium currents appear cephalopod-like in their relative voltage-independence of activation rates, inactivation rates in Strombus are more voltage¬ dependent and thus more similar to typical gastropods. Materials and Methods Force Measurements A strain gauge was used to measure the force exerted by the foot due to electrical stimulation of the motor nerve. Two procedures were used to determine the maximum stimulus frequency that the motor nerve could follow. The first involved varying the time between twin pulses of stimuli and determining the shortest interval which gave summation of the twitch amplitude. The second involved applying a stimulus of a given frequency for - 1 s and monitoring the rate of force development. Similar experiments were conducted on two snails. Cell Preparation and Culture The pedal ganglia was identified and extracted from a dissected snail. The sheath was removed after soaking the ganglia for 45 minutes at room temperature in a 7 mg/mL solution of commercially-available protease IVX (Sigma, St. Louis, MO) dissolved in sea water. Cells from the ganglia were then manually dissociated onto Con A-coated coverslips and cultured at 12 °C for up to a week. Details of the culture medium used are described by Gilly et al. (1990) Recording from Pedal Cells With the use of a List EPC-7 amplifier, conventional whole-cell patch-clamp methods were employed to record Na+ current from pedal neurons. Electrodes made of 7052 glass were pulled and fire-polished so that their resistance in the bath was -0.8-2 M82. Uncompensated input resistance values were typically less than 1 MQ. Series resistance and capacitance compensation were adjusted frequently throughout the course of an experiment by examining the capacitance current transient resulting from an 11 mV test pulse. All recordings were filtered at 5-10 kHz. Linear ionic and capacity currents were removed on-line with a standard P/-4 procedure. External bath recording solutions contained (in mM) 480 Na, 20 MgCl» 10 CaCl», and 20 MgSO, at pH 8.0. Internal pipette recording solutions contained (in mM) 20 tetra methyl ammonium (TMA)-Cl, 80 TMA-glutamate, 50 TMA-F, 10 lysine, 1 EGTA, 1 EDTA, 381 glycine, 291 sucrose, 10 Hepes at pH 7.8 . All recordings analyzed in this study were taken at 22.5 % Determination of Kinetic Parameters Both activation and inactivation parameters were determined by fitting single exponentials to current traces in response to 6 or 12 ms voltage steps, sampled at rates of 10 us and 20 us, respectively. Activation time constants were determined by fitting exponentials to the final rise of lya; i.e., from 3/4 peak amplitude to peak amplitude. Inactivation time constants were determined by fitting lya from the peak to the end of the pulse. Exponentials were not fitted to traces where noise prevented a reasonable fit, as judged by the eye. Characteristics of recovery from inactivation were ascertained by varying the duration and voltage levels between two test voltage pulses of identical amplitude. Results Maximum firing frequency of the motor nerve is - 100 Hz The small size of the Strombus motor nerve prevents easy measurement of action potentials. Maximum firing frequency can be ascertained indirectly, however, by examining the relationship between frequency of motor nerve stimulation and the graded rate of force development by muscles of the foot. Application of a short train (-1 s) of stimulus at various frequencies revealed that the muscle response approached a maximum around à frequency of 100 Hz (Figure 1A). Application of two pulses at various intervals indicated that summation of twitch response did not reliably occur with an interpulse interval of less than about 10 ms (Figure 1B). Thus, both of these experiments indicate that the motor nerve can fire at a maximum frequency of about 100 Hz. Features of Strombus Na- Currents Current-voltage and conductance-voltage relationships Na“ channels of Strombus pedal ganglia cells display typical current-voltage and conductance-voltage relationships (Figure 2). Strombus G-V relationships are similar to those of both cephalopods and gastropods described by Gilly et al. (1997). Like Strombus, Loligo and Aplysia Nat channels, achieve peak conductance around 20 mV and about 2/3 peak conductance at 0 mV. Comparison of Kinetic Parameters for Strombus, Loligo, and Aplysia Nat Current Activation Activation time constants for sodium currents in Strombus pedal cells are much less voltage-dependent than those of slower-moving gastropods, such as Aplysia, as shown in Figure 3A. Furthermore, although Strombus sodium channels activate more slowly than cephalopod channels at all voltages, they display similar exponential voltage dependencies. These distinctions are apparent when one fits single exponential curves of the form Ko + Kjexp(-Kzx) to the Tauon-voltage relationships for Loligo, Strombus, and Aplysia.. Ka values for Loligo, Strombus, and Aplysia sodium channels are .033,.02, and 07 respectively In addition to activation time constants, the time required for the current to reach half its peak value provides a measure of how quickly channels open. As shown in Figure 3B, Strombus sodium channels have slightly longer half-times-to-peak than Loligo sodium channels, but shorter half-times-to-peak than Aplysia sodium channels. These results are consistent with the activation time constant trends in the three organisms; Strombus half-times-to-peak are intermediate between those of Loligo and Aplysia. Unlike the time constant data, however, exponential decay rates for the half-time versus voltage relationships are similar across the genera studied. K» values are .038, .031, and .036 for Aplysia, Strombus, and Loligo, respectively. Inactivation Unlike time constants of activation, the kinetic parameters of inactivation of sodium currents in Strombus pedal ganglia cells display marked voltage-dependence. Strombus cells exhibit fast inactivation rates at high voltages, but much slower inactivation rates at lower voltages (Figure 4). Indeed, inactivation time constants for Strombus sodium channels appear considerably more voltage-dependent than those of both Loligo and Aplysia. Fitting single exponentials to the inactivation-voltage relationships for all three organisms yields K» values of 0.054, 0.116, and 0.015 for Aplysia, Strombus, and Loligo, respectively. Recovery from inactivation Comparative data for recovery from inactivation of sodium channels for cephalopods and gastropods were unavailable for analysis. Across most studied organisms, however, the relationship between the interval between the conditioning and test pulse and relative recovery of Iva is approximately described by an exponential curve Furthermore, recovery from inactivation is highly dependent on the voltage between test pulses and is increasingly rapid at more negative voltage levels (Hille 1992). Strombus pedal ganglia sodium channels clearly exhibit both recovery trends. Recovery is quite rapid at very negative interpulse voltages and less rapid at more positive voltages, as shown in Figure 5A. The relationship between Strombus recovery time constants and voltage is given in Figure 5B. Note that even at an interpulse voltage of -60 mV, Strombus sodium channels fully recover by 10 ms, a value consistent with a maximum motor nerve firing rate of 100 Hz. Toxin sensitivity As shown in Figure 6A, Strombus pedal ganglia sodium channels are resistant to tetrodotoxin (TTX), a trait they share with many other gastropods (Adams et al. 1980). In contrast, cephalopod sodium channels, like those most other organisms, are highly sensitive to TTX and would be completely blocked at the concentration used. Strombus sodium channels also display little sensitivity to Conus californicus milked venom (Figure 6B), which also is known to block cephalopod sodium channels at the tested concentration (Gilly, unpublished results). Discussion Results described in this paper are consistent with the hypothesis that Strombus motor neurons can transmit high frequency information in order to control their rapid escape response. Less clear, however, is the extent to which biophysical properties of Strombus sodium channels deviate from those of the typical gastropod and resemble those of the typical cephalopod. Although the voltage-dependence of activation time constants is similar in Strombus and Loligo, Loligo channels are considerably faster at all voltages. Furthermore, recordings from Loligo giant fiber lobe cells were taken at 10 degrees, whereas recordings from Strombus pedal cells were taken at 22.5 degrees. Because channel kinetics generally become more rapid as the temperature increases, the relative slowness of Strombus sodium channels might be taken as an argument against their truly being "cephalopod like." However, activation time constants in Strombus may be comparable to those of cephalopods when recordings are made at temperatures more representative of their natural tropical environment (i.e. -30 degrees). Further experiments at ecologically relevant temperatures are necessary to confirm this hypothesis. Ultimately, because the absolute speed of channel kinetics is so dependent on temperature, what is most relevant is that Strombus sodium channels display cephalopod¬ like voltage-activation trends. Inactivation-voltage relationships in Strombus are even more difficult to interpret. The same cell whose activation time constants displayed relatively little voltage- dependence demonstrated highly voltage-dependent inactivation time constants. Similar trends were evident in multiple Strombus pedal cells. Given that activation and inactivation gating is thought to be coupled in many organisms, including squid and mammals, the trends observed in Strombus are somewhat surprising. It is possible that Strombus Na+ channels are true deviants, activating in a voltage-independent fashion, but inactivating in a voltage-dependent fashion. Strombus Kinetic parameters described in this paper may be confounded by the fact that exponentials were fit to currents that contained both inactivating and non¬ inactivating components. In their comparative analysis of gastropod and mollusk Na¬ channels, Gilly et al. fit exponentials only to the inactivating current component, isolated by a prepulse subtraction method (see paper for details). Similar methods were not employed in the study of Strombus Na+ currents, however, because isolated fast- inactivating traces are much noisier than non-subtracted traces, preventing reliable fitting of kinetic parameters. While the non-inactivating current seen in Strombus pedal cells was generally quite small (on the order of -200 pA at 0 mV), it is possible that failing to subtract this current component distorted the estimations of activation and inactivation time constants. Despite an indication that Strombus sodium channels resemble cephalopod channels in their voltage-dependence of activation, the data on toxin effects suggests a fundamental dissimilarity in structure. Even if Strombus has evolved sodium channels that open quickly at middle voltage levels-an adaptation which would enable their rapid escape-it seems quite reasonable that structural features of gastropod channels independent of activation and inactivation gating have been preserved. Conclusion This preliminary analysis of Strombus sodium channels suggests that voltage-dependent features of activation and inactivation differ from those of both cephalopods and other gastropods. Further confirmation of the apparent lack of coupling between activation and inactivation in Strombus are warranted, and should be achieved ideally by fitting exponentials to prepulse-subtracted traces. If the observed trends are replicated, Strombus sodium channels deserve much closer biophysical analysis. Acknowledgments This project could not have been completed without the invaluable help and guidance of my advisor, William Gilly. Thank you also to Rob DeConde for his commitment to acquiring excellent cells. Td also like to thank all of the members of the Gilly lab who provided patience answers to my many questions. Literature Cited Adams, D. J., Smith, S. J, and Thompson, S.H. lonic currents in molluskan soma. Annu. Rev. Neuroscien. 3: 141-167, 1980. Berg, Carl J. A Comparative Ethological Study of Strombid Gastropods. Behavior: 274- 321, 1974. Gilly, W. F. Lucero, M. T., and Horrigan, F. T. Control of the spatial distribution of sodium channels in giant fiber lobe neurons of the squid. Neuron 5: 663-674, 1990. Gilly, W. F., Gillette, R. and McFarlane, M. Fast and Slow Na Channels in Molluscan Neurons. J. Neurophysiol. 77: 2373-2384, 1997 Hille, Bertil. Ionic Channels of Excitable Membranes. Sunderland, Mass: Sinauer Associates, 1992. Figure Legend Figure 1 A: Relationship between frequency of stimulus applied to the Strombus motor nerve and the maximum rate of force development in the foot. B: Relationship between the duration between twin stimuli to the Strombus motor nerve and the amplitude of force exerted by the foot. Figure 2 A: Instantaneous I-V curve for sodium current in Strombus pedal ganglia cells. B: Conductance-voltage relationship for Strombus pedal ganglia cells, determined from the equation G-(Vmembrane-Vreversal). The reversal potential was -85 mV, determined by extrapolating the IV curve in A to where it intersects the x-axis. Figure 3 A: Voltage-dependence of activation time constants in Strombus pedal cells, Loligo giant fiber cells, and Aplysia pedal cells. B: Voltage-dependence of half-times-to-peak in Strombus, Loligo, and Aplysia. Figure 4 Voltage-dependence of inactivation time constants in Strombus, Loligo, and Aplysia. Figure 5 A: Recovery from inactivation in Strombus pedal cells. The different traces represent different interpulse voltage levels. B: Dependence of the time constants of the exponentials fitted to the recovery curves in 5A on interpulse voltage levels. Figure 6 A: Effect of 200 nM TTX on Strombus Nat channels. B: Effect of 1:100 dilution of Conus californicus milked venom on Strombus Na¬ channels. Figure 1 22.5- 2.0 - 1.5 - 1.0- 2 60x10° - 50 40 30 20 X 100 interpulse duration 40 frequency of stimulus (Hz) 150 20 100 Figure 2 B -20 50 -500 + 2-1000 2 1500 -2000 -2500- 0.8 0.6 0.2 0— 10 Sheet4 40 20 60 voltage 10 30 50 70 90 L voltage Page 1 1200- 1000: 800 - 600- 400- 200 - 14— 1.2- 1.0- 108 0.6 - 0.4- A Strombus pedal cells □ Aplysia pedal cells Loligo giant fiber lobe cells — S -20 40 60 voltage (mV) Loligo giant fiber lobe cells □ Aplysia pedal cells A Strombus pedal cells — 20 40 voltage (mV) Figure 3 Figure 4 4 3- 2- 1— □ □ Aplysia pedal cells Loligo giant fiber lobe cells strombus pedal cells — A 60 20 voltage (mV) -20 1.0- 0.8- 0.6 - 0.4 0.2- 2.5 - 2.0 - 1.5 - 1.0 - -100 duration between test pulses (ms) -80 -70 interpulse voltage (mV) -60 + -60 mV A -70 mV -80 mV □ -90 mV • -100 mV Figure 5 Figure 6 o after application of TIX -200 - -400 - before application of TIX -600 — O 2 4 10 12 14 time (ms) after application of Conus venom M -200 - 400 - -600 - -800 - before application of Conus venom -1000 - -1200 - 2 3 6 7 time (ms)