Voltage-dependent inhibition of rat skeletal muscle sodium channels by aminoglycoside antibiotics

Abstract

Aminoglycoside (AG) antibiotics interact with numerous biological molecules, including some voltage-gated ion channels. The present study demonstrates that 4,5-disubstituted (neomycin class) and 4,6-disubstituted (kanamycin class) AGs inhibit whole-cell currents through cloned rat skeletal muscle sodium channels (mu1, Na(V)4.1). Increases in the amplitude of the step command reduced inhibition by extracellular AGs but increased inhibition by intracellularly applied AGs, indicating that the block was voltage dependent. Furthermore, intracellular neamine or sisomycin hastened the rate of macroscopic current decay at positive voltages. Extracellular solution containing sodium ions slowed the rate of current decay in the presence of intracellular sisomycin and decreased the apparent affinity of sisomycin from the intracellular side twofold. Current inhibition by extracellularly or intracellularly applied AGs was well fitted by the Woodhull model of pore block. The model indicated that most extracellularly applied AGs interact at a site that is an electrical distance of approximately 10-15% from the outside, whereas intracellularly applied neamine or sisomycin bind to sites that are approximately 49% and approximately 24%, respectively, into the electric field from the inside. Our data suggested that AG antibiotics induce a low-affinity, voltage-dependent block of mu1 channels.

On the Interaction of Neomycin with the Slow Vacuolar Channel ofArabidopsis thaliana

“…Inhibition of SV macroscopic currents depended on the concentration of neomycin and the applied membrane potential. Dose-response analyses of these data suggested a voltage-dependent Hill constant K h and a 1:1 stoichiometry of the interaction between neomycin and the channel protein, which is not surprising in consideration of the relative large size of the neomycin molecule (MW = 615) and was also reported in the interaction of neomycin with animal sodium channels (Yeiser et al, 2004) and L-type calcium channels (Haws et al, 1996). At the single channel level, neomycin at a concentration of 50 μM caused a voltageindependent reduction of the channel conductance at negative membrane potentials.…”

“…The reduction of single channel conductance increased dramatically at progressively more positive potentials (Fig. 7 B); this provides evidence for a pore block due to binding of positively charged amino groups of the neomycin molecule at a site located within the electrical fi eld of the ionconducting pathway, as described for neomycin block of other cation channels (Nomura et al, 1990;Haws et al, 1996;Winegar et al, 1996;Mead and Williams, 2002;Yeiser et al, 2004). Indeed, the much slower channel deactivation at negative membrane potentials in the presence of neomycin (Fig.…”

“…In contrast to neomycin B, kanamycin A is an antibiotic of the 4,6-disubstituted aminoglycoside subclass ( Fig. 1 ), with only four amine groups and a net charge of +1.7 at pH 7.4 ( Yeiser et al, 2004 ). Applied at a concentration of 1 mM to the bath solution, kanamycin had a stimulatory effect on macroscopic SV currents at membrane potentials <+80 mV, which progressively decreased and eventually turned into an inhibitory effect with increasing voltage ( Fig.…”

“…An analogous relationship between the number of amine groups of aminoglycosides and their blocking potency was postulated for animal Ca 2+ -activated potassium channels (Nomura et al, 1990) and L-type calcium channels (Haws et al, 1996). However, no such correlation was found for sodium channels (Yeiser et al, 2004), favoring alternative hypotheses on the binding of aminoglycosides based on the size of the molecule or on the location of the amine groups.…”

“…In cell biology, neomycin is widely used in studies on inositol 1,4,5-triphosphate (IP 3 )-dependent intracellular calcium release, as it inhibits the IP 3 -creating enzyme PLC (Downes and Michell, 1981;Gabev et al, 1989). Furthermore, neomycin was shown to block many types of animal ion channels: Ca 2+ -activated potassium channels from rat brain (Nomura et al, 1990), mechanosensitive (Winegar et al, 1996) and L-type calcium channels (Haws et al, 1996) from mouse skeletal muscle, P/Q-type calcium channels of guinea pig brain (Pichler et al, 1996), nicotinic acetylcholine receptor channels (Rothlin et al, 2000;Shi et al, 2002), the cardiac ryanodine receptor channel (Mead and Williams, 2002;Mead and Williams, 2004), rat skeletal muscle sodium channels (Yeiser et al, 2004), and neuronal vanilloid receptor channels (Raisinghani and Premkumar, 2005). Interestingly, aminoglycosides can also have activating properties, e.g., on neuronal NMDA receptors (Lu et al, 1998).…”