Open-channel block of Na+ channels by intracellular Mg2+ |
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Authors: | M. Pusch |
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Affiliation: | (1) Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-3400 Göttingen, Federal Republic of Germany;(2) Present address: Istituto di Cibernetica e Biofisica, CNR, Via Dodecaneso 33, I-16146 Genova, Italy |
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Abstract: | 1. Macroscopic and single-channel currents through several types of cloned rat brain Na+ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na+ channels in chromaffin cells, intracellular Mg2+ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na+ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg2+. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg2+ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na]i); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg2+ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg]i. This indicates that Mg2+ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 108 M–1 s–1 and 3.6 · 105 s–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg2+ strongly depends on [Na]i. This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg2+ block at physiological levels of [Mg]i.abbreviations [Na]i intracellular Na+ concentration - [K]i intracellular K+ concentration - [Mg]i intracellular Mg2+ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium |
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Keywords: | Sodium channel Mg2+ block Open-channel noise Patch clamp |
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