Cell membrane sodium channel activation

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

A medium throughput approach to evaluating sodium channel activity is the measurement of sodium flux across cell membranes [103]. In these experiments, a tracer that permeates the channel and is easily quantifiable is used to analyze sodium influx. Traditionally, radioactive tracers such as 22Na+ or [14C]guanidinium have been used. Alternatively, Li+ can be used as a tracer and analyzed by atomic absorption spectrometry. Sodium flux assays can be used to test approximately 105 compounds per year. They offer a robust readout of channel activity, but lack voltage control and temporal resolution. To examine sodium channel blockade by measuring sodium flux,... 

A dramatic increase in Na"" permeability requires a dramatic increase in the number of channels that allow Na"" to enter the cell. Thus, the resting p is only a small frac tion of what it could be because most membrane sodium channels are closed at rest. What stimulus causes the hidden Na" channels to reveal themselves It turns out that the activation of these Na channels is triggered by membrane depolarization. When is at its usual resting levels around -70 mV, these Na" channels are closed and p is low. However, depolarization causes the channels to open. Because the voltage-activated Na channels respond to depolarization, the response of the membrane to depolarization is regenerative, and thus explosive (Figure 10.3). A small depolarization of the membrane opens Na" channels, which causes influx of Na"" into the cell and additional depolarization, which in turn opens more Na" channels. This explains the all-or-none nature of the action potential once it is triggered, it runs to completion. 

A variety of in vitro assays have been developed that minimize or avoid live animal experimentation. Several capitalize on the sodium channel s affinity for these toxins. Neuronal cell lines lyse in the presence of veratridine, a sodium channel activator, and ouabain, which prevents removal of the excessive sodium ions allowed in by veratridine. In the presence of both these drugs, a sodium channel blocker such as saxitoxin rescues the cells. Cellular viability can then be measured by adding tetrazolium salts that are metabolized by living cells to a colored product. Alternatively, isolated cellular membranes, typically from brain tissue, are used to bind radiolabeled saxitoxin. After incubating receptor and radioligand in the presence of a test sample, any radiolabeled saxitoxin bound to the cell membranes are deposited onto filters by vacuum pressure. Radioactivity from the labeled saxitoxin is then measured with a signal reduction indicating the presence of saxitoxin. 

Bettendorlf, L., and Wins, P., 1994. Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP. Journal of Biological Chemistry. 269 14379-14385. 

Additional cellular events linked to the activity of blood pressure regulating substances involve membrane sodium transport mechanisms Na+/K.+ ATPase Na+fLi countertransport Na+ -H exchange Na+-Ca2+ exchange Na+-K+ 2C1 transport passive Na+ transport potassium channels cell volume and intracellular pH changes and calcium channels. 

Ion channels are large proteins which form pores through the neuronal membrane. The precise structure and function of the ion channels depend on their physiological function and distribution along the dendrites and cell body. These include specialized neurotransmitter-sensitive receptor channels. In addition, some ion channels are activated by specific metal ions such as sodium or calcium. The structure of the voltage-dependent sodium channel has been shown to consist of a complex protein with both a hydrophilic and a hydrophobic domain, the former domain occurring within the neuronal membrane while the latter domain occurs both inside and outside the neuronal membrane. 

Figure 2.2 Diagram of a voltage-activated sodium channel protein. The channel is composed of a long chain of amino acids intercormected by peptide bonds. The amino acids perform specific functions within the ion channel. The cylinders represent amino acid assemblies located within the membrane of the nerve cell and responsible for the foundation of the ion pore.

Neurotransmitters can either excite or inhibit the activity of a cell with which they are in contact. When an excitatory transmitter such as acetylcholine, or an inhibitory transmitter such as GABA, is released from a nerve terminal it diffuses across the synaptic cleft to the postsynaptic membrane, where it activates the receptor site. Some receptors, such as the nicotinic receptor, are directly linked to sodium ion channels, so that when acetylcholine stimulates the nicotinic receptor, the ion channel opens to allow an exchange of sodium and potassium ions across the nerve membrane. Such receptors are called ionotropic receptors. 

Lidocaine is the most widely used local anesthetic. Its excellent therapeutic activity is fast-acting and lasts sufficiently long to make it suitable for practically any clinical use. It stabilizes cell membranes, blocks sodium channels, facilitates the secretion of potassium ions out of the cell, and speeds up the repolarization process in the cell membrane. It is used for terminal infiltration, block, epidural, and spinal anesthesia during operational interventions in dentistry, otolaryngology, obstetrics, and gynecology. It is also used for premature ventricular extrasystole and tachycardia, especially in the acute phase of cardiac infarction. Synonyms for this drug are xylocaine, neflurane, and many others. 

Figure 12.6 Mechanism of action of mineralocortjcoid receptor antagonists in the collecting tubule. Aldosterone enters the tubular cell by the basolateral surface and binds to a specific mineralocorticoid receptor (MNR) in the cytoplasm. The hormone receptor complex triggers the production of an aldosterone-induced protein (AlP) by the cell nucleus (NUC). The AIP acts on the sodium ion channel (ic) to augment the transport of Na+across the basolateral membrane and in to the cell. An increase in AIP activity leads to the recruitment of dormant sodium ion channels and Na pumps (P) in the cell membrane. AIP also leads to the synthesis of new channels and pumps within the cell. The increase in Na+conductance causes electrical changes in the luminal membrane that favour the excretion of intracellular cations, such as K+and H-h. Spironolactone competes with aldosterone for the binding site on the MNR and forms a complex which does not excite the production of AIP by the nucleus.

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