Identification of two sodium channel subtypes in chick heart and brain (original) (raw)
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Journal of General Physiology, 1995
Tyrosine 401 of the skeletal muscle isoform (0`1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn ~+ and Cd 2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na § current for a series of point mutations at residue Tyr401 of the rat 0`1 Na channel expressed in Xen0pus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na § currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with/~ values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7-and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn 2+ (/~ = 8-23 0`M) required Cys, His, or Asp, three residues commonly found to coordinate direcdy with Zn 2+ in metalloproteins. For the Cys mutant of 0.1 and also for the cardiac isoform Na channel (rhl) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na § conductance by Zn ~ § reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The ASp mutant also displayed enhanced affinity for inhibition of conductance by Ca 2+ (/~ = 0.3 mM vs ~40 mM in wild type), but block by Ca ~ § was incomplete, saturating at ~69% inhibition. In contrast, Cd 2 § completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at
The Journal of Physiology, 1977
1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal frog muscle, has been measured. 2. A saturable component of saxitoxin binding, which was inhibited by tetrodotoxin, was detected in all preparations, as well as a component of non-saturable binding. The values for the maximum saturable capacity, M, and the equilibrium binding constant, K, for normal rat diaphragm muscle were: M = 24-4 f-mole. mg wet-L, and K = 3-8 nM. 3. Denervation of rat diaphragm muscle reduced the maximum binding capacity per unit weight .to 16-5 f-mole. mg-'.tThe value of K remained virtually unchanged at 4-2 nM. 4. It is suggested that the decrease in density per unit weight does not reflect any change in the density of sodium channels per unit area of membrane. 5. Two varieties of the same species of frog, Rana pipiens, were examined. In one variety (Southern) the value of M was 25-6 f-mole. mg-' and the value of K was 4-3 nM. In the Northern variety the maximum binding capacity was less, M being 14-6 f-mole. mg-'; the value of K was 3-8 nM.
Biochemistry, 1987
The single-channel blocking kinetics of tetrodotoxin (TTX), saxitoxin (STX), and several STX derivatives were measured for various Na-channel subtypes incorporated into planar lipid bilayers in the presence of batrachotoxin. The subtypes studied include N a channels from rat skeletal muscle and rat brain, which have high affinity for TTX/STX, and N a channels from denervated rat skeletal muscle and canine heart, which have about 20-60-fold lower affinity for these toxins at 22 O C . The equilibrium dissociation constant of toxin binding is an exponential function of voltage (e-fold per 40 mV) in the range of -60 to +60 mV. This voltage dependence is similar for all channel subtypes and toxins, indicating that this property
Biophysical Journal, 1998
The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na ϩ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by Kirsch et al. (1994. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na ϩ channel isoform (I), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp 400 , Glu 755 , and Lys 1237 , all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu 758 and Asp 1532. On the other hand, mutation of Tyr 401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na ϩ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr 401 , and STX interacts more strongly with the more extracellular residues.
Proceedings of the National Academy of Sciences, 1980
The saxitoxin-binding component (SBC) of the excitable membrane sodium channel has been solubilized and purified from rat skeletal muscle sarcolemma. Phospholipid was required in mixed micelles with detergent for stability of the mammalian SBC. Even at optimal detergent-to-phospholipid ratio, the solubilized SBC showed significant temperature-dependent loss of specific toxin binding with time, necessitating maintenance of low temperatures during purification. Characteristics of saxitoxin binding to the solubilized material closely resembled those seen in intact membranes. A weak anion-exchange column was synthesized; it provided rapid 10- to 20-fold purification of the solubilized SBC. Additional necessary purification was obtained by chromatography on immobilized wheat germ agglutinin. Specific saxitoxin-binding activity of the purified material averaged approximately 1500 pmol of saxitoxin bound per mg of protein. Three bands were present in this material on sodium dodecyl sulfate...
Proceedings of The National Academy of Sciences, 1986
The effect of two pL-conotoxin peptides on the specific binding of [3H]saxitoxin was examined in isolated plasma membranes of various excitable tissues. pt-Conotoxins GITIA and GIHIB inhibit [3H]saxitoxin binding inlEkctrophorus electric organ membranes with similar Kds of %50 x 10-9 M in a manner consistent with direct competition for a common binding site. GITIA and GIIIB similarly compete with the majority (80-95%) of [3Hlsaxitoxin binding sites in rat skeletal muscle with Kds of -25 and "140 x 10-9 M, respectively. However, the high-affinity saxitoxin sites in lobster axons, rat
Biophysical Journal, 1994
Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat Ila co-expressed with ,B1 subunit, Br2a + p1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + (1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.
Animal Toxins Influence Voltage-Gated Sodium Channel Function
Handbook of Experimental Pharmacology, 2014
Voltage-gated sodium (Nav) channels are essential contributors to neuronal excitability, making them the most commonly targeted ion channel family by toxins found in animal venoms. These molecules can be used to probe the functional aspects of Nav channels on a molecular level and to explore their physiological role in normal and diseased tissues. This chapter summarizes our existing knowledge of the mechanisms by which animal toxins influence Nav channels as well as their potential application in designing therapeutic drugs.