Abstract
Isolated Na currents were studied in cultured chick sensory neurons using the patch clamp technique. On membrane depolarization, whole cell currents showed the typical transient and voltage-dependent time course as in nerve fibres. Na currents appeared at about-40 mV and reached maximum amplitude at around-10 mV. At low voltages (-30 to 0 mV), their turning-on was sigmoidal and inactivation developed exponentially. The ratio of inactivation time constants was found to be smaller than in squid axons and comparable to that of mammalian nodes of Ranvier. Peak conductance and steady-state inactivation were strongly voltage-dependent, with maximum slopes at-17 and-40 mV, respectively. The reversal potential was close to the Nernst equilibrium potential, indicating a high degree of ion-selectivity for the channel. Addition of 3μM TTX, or replacement of Na by Choline in the external bath, abolished these currents. Internal pronase (1 mg/ml) and N-bromoacetamide (0.4 mM) made inactivation incomplete, with little effect on its rate of decay.
Single Na channel currents were studied in outside-out membrane patches, at potentials between-50 and-20 mV. Their activation required large negative holding potentials (-90 mV). They were fully blocked by addition of TTX (3 μM) to the external bath. At-40 mV their mean open time was about 2ms and the amplitude distribution could be fitted by a single Gaussian curve, indicating the presence of a homogeneous population of channels with a conductance of 11±2 pS. Probability of opening increased and latency to first opening decreased with increasing depolarization. Inactivation of the channel became faster with stronger depolarizations, as measured from the inactivation time course of sample averages. Internal pronase (0.1 mg/ml) produced effects on inactivation comparable to those on whole cell currents. Openings of the channel had a tendency to occur in bursts and showed little inactivation during pulses of 250 ms duration. The open lifetime of the channel at low potentials (-50,-40 mV) was only three times larger than in control patches, suggesting that Na channels in chick sensory neurons can close several times before entering an inactivating absorbing state.
Similar content being viewed by others
References
Adams DJ, Gage PW (1979) Characteristics of sodium and calcium conductance changes produced by membrane depolarization in an Aplysia neurone. J Physiol (Lond) 289:143–161
Aldrich RW, Corey DP, Stevens CF (1983) A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature 306:436–441
Almers W, McCleskey EW (1984) Non selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. J Physiol (Lond) 353:585–628
Armstrong CM (1981) Sodium channels gating currents. Physiol Rev 61:644–683
Armstrong CM, Bezanilla F, Rojas E (1973) Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol 62:375–391
Baccaglini PI (1978) Action potentials of embryonic dorsal root ganglion neurons in Xenopus tadpoles. J Physiol (Lond) 283:585–604
Barde YA, Edgar D, Thoenen H (1980) Sensory neurons in culture: changing requirements for survival factors during embryonic development. Proc Natl Acad Sci USA 77:1199–1203
Brodwick MS, Eaton DC (1978) Sodium channel inactivation in squid axon is removed by high internal pH or tyroxine-specific reagents. Science 200:1494–1496
Carbone E, Lux HD (1984a) A low voltage-activated calcium conductance in embryonic chick sensory neurons. Biophys J 46:413–418
Carbone E, Lux HD (1984b) A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurons. Nature 310:501–502
Carbone E, Lux HD (1984c) Single Na and Ca channels in vertebrate sensory neurons. Int. Congr. Biophys Abstr (Bristol) 1:272
Carbone E, Testa PL, Wanke E (1981) Intracellular pH and ionic channels in the Loligo vulgaris giant axon. Biophys J 35:393–413
Chachelin AB, De Peyer JE, Kokubun S, Reuter H (1983) Sodium channels in cultured cardiac cells. J Physiol (Lond) 340:389–401
Chandler WK, Meves H (1970a) Evidence for two types of sodium conductance in axons perfused with sodium fluoride solutions. J Physiol (Lond) 211:653–678
Chandler WK, Meves H (1970b) Slow changes in membrane permeability and longlasting action potentials in axons perfused with fluoride solutions. J Physiol (Lond) 211:707–728
Chiu SY, Ritchie JM, Rogart RB, Stagg D (1979) A quantitative description of membrane currents in rabbit myelinated nerve. J Physiol (Lond) 292:149–166
Conti F, DeFelice LJ, Wanke E (1975) Potassium and sodium ion current noise in the membrane of the squid giant axon. J Physiol. (Lond) 248:45–82
Conti F, Hille B, Neumcke B, Nonner W, Stämpfli R (1976) Measurement of the conductance of the sodium channel from current fluctuations at the node of Ranvier. J Physiol (Lond) 262:699–727
Cuervo LA, Adelman WJ (1970) Equilibrium and kinetic properties of the interaction between tetrodotoxin and the excitable membrane of the squid giant axon. J Gen Physiol 55:309–335
Dichter MA, Fischbach GD (1977) The action potential of chick dorsal root ganglion neurones maintained in cell culture. J Physiol (Lond) 267:281–298
Dodge FA, Frankenhäuser B (1959) Sodium currents in the myelinated nerve fibre of Xenopus Laevis investigated with the voltage clamp technique. J Physiol (Lond) 148:188–200
Dubinsky JM, Oxford GS (1984) Ionic currents in two strains of rat anterior pituitary tumor cells. J Gen Physiol 83:309–339
Dunlap K, Fischbach GD (1981) Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurons. J Physiol (Lond) 317:519–535
Fenwick EM, Marty A, Neher E (1982) Sodium and calcium channels in bovine chromaffin cells. J Physiol (Lond) 331:599–635
Fernandez JM, Fox AP, Krasne S (1984) Membrane patches and whole cell membranes: a comparison of electrical properties in rat clonal pituitary (GH3) cells. J Physiol (Lond) 356:565–585
Fox JM (1976) Ultra-slow inactivation of the ionic currents through the membrane of the myelinated nerve. Biochim Biophys Acta 426:245–257
Frankenhäuser B (1963) Inactivation of the sodium-carrying mechanism in myelinated nerve fibres of Xenopus Laevis. J Physiol (Lond) 169:445–451
French RJ, Horn R (1983) Sodium channel gating: Models, Mimics and Modifiers. Annu Rev Biophys Bioeng 12: 319–356
Goldman L, Schauf CL (1973) Quantitative description of sodium and potassium currents and computed action potentials in Myxicola giant axons. J Gen Physiol 61:361–384
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100
Hess P, Tsien RW (1984) Mechanisms of ion permeation through calcium channels. Nature (Lond) 309:453–456
Hodgkin AL, Huxley AF (1952a) The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J Physiol (Lond) 116:497–506
Hodgkin AL, Huxley AF (1952b) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol (Lond) 117:500–544
Horn R, Lange K (1983) Estimating kinetics constants from single channel data. Biophys J 43:207–223
Horn R, Vandenberg CA (1984) Statistical properties of single sodium channels. J Gen Physiol 84:505–534
Horn R, Vandenberg CA, Lange K (1984) Statistical analysis of single Na channels. Effects of N-Bromoacetamide. Biophys J 45:323–335
Ishizuka S, Hattori K, Akaike (1984) Separation of ionic currents in the somatic membrane of frog sensory neurons. J Membr Biol 78:19–28
Kameyama M (1983) Ionic currents in cultured dorsal root ganglion cells from adult guinea pigs. J Membr Biol 72:195–203
Kostyuk PG, Veselovsky NS, Tsyndrenko AY (1981) Ionic currents in the somatic membrane of rat dorsal root ganglion neurons. I. Sodium currents. Neuroscience 6:2423–2430
Kostyuk PG, Veselovsky NS, Fedulova SA (1981) Ionic currents in the somatic membrane of rat dorsal root ganglion neurons. II. Calcium currents. Neuroscience 6:2431–2437
Kunze DL, Lacerda AE, Wilson DL, Brown AM (1985) Reopening, waiting and inactivating properties of single Na channels. J Gen Physiol (in press)
Lux HD, Brown AM (1984) Patch and whole cell calcium currents recorded simultaneously in snail neurons. J Gen Physiol 83:727–750
Matteson DR, Armstrong CM (1984) Na and Ca channels in a transformed line of anterior pituitary cells. J Gen Physiol 83:371–394
Meves H (1978) Inactivation of the sodium permeability in squid giant fibres. Prog Biophys Mol Biol 33:207–230
Moolenaar WH, Spector I (1978) Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions. J Physiol (Lond) 278:265–286
Mudge AW, Leeman SE, Fischbach GD (1979) Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration. Proc Natl Acad Sci USA 76:526–530
Nagy K, Kiss T, Hof D (1983) Single Na channels in mouse neuroblastoma cell membrane. Indications for two open states. Pflügers Arch 399:302–308
Otsuka M, Konishi S (1976) Substance P and excitatory transmitter of primary sensory neurons. Cold Spring Harbor Symp Quant Biol 40:135–143
Oxford GS, Wu CH, Narahashi T (1978) Removal of sodium channel inactivation in squid giant axons by N-bromoacetamide. J Gen Physiol 71:227–247
Patlak J, Horn R (1982) Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches. J Gen Physiol 79:331–351
Patlak JB, Ortiz M (1985) Slow currents through single sodium channels of the adult rat heart. J Gen Physiol 86:89–104
Schwarz IR, Ulbricht W, Wagner HH (1973) The rate of action of tetrodotoxin on myelinated nerve fibres of Xenopus Laevis and Rana Esculenta. J Physiol (Lond) 233:167–194
Sigworth FJ, Neher E (1980) Single Na+ channel currents observed in cultured rat muscle cells. Nature (Lond) 287:447–449
Swenson RP (1980) Gating charge immobilization and sodium current inactivation in internally perfused crayfish giant axons. Nature (Lond) 287:644–645
Vandenberg CA, Horn R (1984) Inactivation viewed through single sodium channels. J Gen Physiol 84:535–564
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Carbone, E., Lux, H.D. Sodium channels in cultured chick dorsal root ganglion neurons. Eur Biophys J 13, 259–271 (1986). https://doi.org/10.1007/BF00254208
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00254208