Caracterización electrofisiológica y farmacológica de los canales de calcio voltaje dependientes de los terminales motores del ratón

Calcium has been identified in a wide variety of biological systems as a second messenger. It has been demonstrated that it has a key role in different processes such as excitation-contraction coupling, neurotransmitter and hormone release, gene activation, some mechanisms associated with learning and memory and many other physiological processes. According to the vesicular hypothesis, Ca²+ entry into the presynaptic nerve terminals is a pre-requisite for transmitter release. It has been shown that Ca²+ fluxes are activated by depolarization and that the amount of transmitter release is dependent on the level of intracellular calcium concentration. This increase in Cap concentration is achieved by the opening of voltage dependent calcium channels (VDCC) after the arrival of the action potential to the nerve terminal. The neuromuscular junction and the squid giant synapse have proved to be excellent preparations to study the sequence of events that take place during synaptic transmission and are a model for understanding the synaptic mechanisms involved in chemical synapses throughout the animal kingdom. Different types of calcium channels have been described, according to their biophysical and pharmacological properties. Llinas y Yarom (l981) demonstrated the coexistence of at least two different types of calcium channels, being one of them of low threshold, LVA low voltage activated- and the other of high threshold HVA - high voltage activated. Later, the presence of these different calcium currents were shown in chicken DRG (dorsal root ganglion) neurones. LVA currents are activated by weak depolarizations from negative potentials and decay rapidly after their activation. HVA currents are activated from more positive holding potentials, and inactivate slowly. The LVA channels are known as T-type calcium channels due to their transient activation. The HVA channels include a great number of subtypes. At least four different types of HVA channels have been distinguished at nerve cells. The L-type calcium channel is sensitive to a family of organic compounds, the dihydropyridines (DHP). There are different DHP, some of them are agonists and others antagonists of the calcium channels. The N-type calcium channels were first described as sensitive to a toxin derived from a marine snail, omega-conotoxin GVIA (ω-CgTx GVIA). There are many other polipeptides obtained from snail and spider venoms which block N-type calcium channels, however, ω-CgTx GVIA is the most wider used. The P-type calcium channel, was initially described in cerebellar Purkinje cells, it was found to be insensitive to DHP and ω-CgTx GVIA, but was potently blocked by a low molecular weight fraction (FTX) and a polypeptide (ω-Aga-IVA), both toxins purified from the venom of the funnel-web spider Agelenopsis aparta. More recently the Q-type calcium channel was described in cerebellar granule cells. This new type of channel is insensitive to DHP and ω-CgTx GVIA, but sensitive to high concentrations of ω-Aga-IVA (hundreds of nanomolar), and also blocked by a polypeptide purified from the snail venom, ω-CgTx M-VIIC. All these different channels, may coexist at a neuronal soma and also at the nerve terminals. However, in some preparations the combined use of all the calcium channel blockers, cannot suppress Ca²+ currents completely, indicating the existence of still unidentified VDCC. Increasing information about VDCC is coming from the field of molecular biology. Molecular cloning of genes which code for the al subunit of VDCC, provided information about the structure of this transmembrane proteins, and the expression of its products in Xenopus oocytes made their biophysical and pharmacological characterization possible. Although it is possible to establish a relationship between the product of the genes and calcium channels characterized ‘in situ', in many cases there is no good correlation between the properties of the expressed calcium channels and those studied in situ. The existence of different VDCC, made it interesting to explore about their specific function and whether a particular type was responsible for synaptic transmission. Electrophysiological recording is the most confident technique to study the channels which take part in synaptic transmission. In 1984, Kerr and Yoshikami showed that frog neuromuscular transmission was abolished by ω-CgTx GVIA, which acted by blocking Ca²+ entry into the presynaptic terminal. In contrast, ω-CgTx GVIA does not produce any effect in mammalian synaptic transmission electrically evoked in normal conditions. In the central nervous system (CNS), depending on the structure, it was shown that different VDCC subtypes are involved. At the neurosecretory terminals of the neurohypophysis it seems that both L- and N- type VDCC mediate neurosecretion (Lemos and Nowicky, 1989). Takahashi and Momiyama (1993) showed that in three different areas of the CNS, P-like and N-type VDCC are involved to varying degrees in synaptic transmission, while L-type seems not to be related with transmitter release. In the synapse between hipocampal CA3 and CAI neurons, transmitter release is mediated by N-type calcium channels and other type of channels, whose pharmacology resembles Q-type calcium channels. Stanley (1991) determined that transmitter release at the calyx synapse of chicken cilliary ganglion is due to Ca²+ flux through N-type calcium channels It is also possible to have an approach about the VDCC involved in transmitter release, by means of colorimetric and biochemical techniques. Yawoo et al. showed that Ca²+ concentration was diminished in the presence of ω-CgTx GVIA, from what derives that N-type calcium channels mediate calcium entry at the chick cilliary ganglion synapse. Many groups showed that K+ evoked neurotransmitter release in CNS slices, is inhibited by coCng GVIA as well as by ω-Aga-IVA, depending on the neurotransmitter and the area studied (Turner et al., 1992; Kimura et al., 1994). At the mammalian neuromuscular junction, under normal conditions, transmitter release is resistant to DHP and to ω-CgTx GVIA, indicating that neither L nor N-type VDCC are mediating transmitter release. However, in cut muscle fibres it was reported that L-type channels antagonists diminish quantal content, when release is previously stimulated by the L-type channel agonist BayK 8644 (Atchinson, 1987). It was also reported that ω-CgTx GVIA prevents the facilitatory effects of noradrenaline on the evoked release of [³H] Acetylcholine from mammalian motor nerve terminals (Wessler et al., 1990).Fil:Protti, Darío Alejandro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina