Gentamicin blocks ACh-evoked K+ current in guinea-pig outer hair cells by impairing Ca2+ entry at the cholinergic receptor.
Aminoglycoside antibiotics such as gentamicin are known to block the medial olivocochlear efferent system. In order to determine whether this inhibition takes place at the postsynaptic cholinergic receptors in outer hair cells (OHCs), we studied the effects of these polycationic molecules on cholinergic currents evoked in isolated guinea-pig OHCs. The cholinergic response of OHCs involves nicotinic-like receptors (nAChRs) permeable to Ca2+ ions that activate nearby Ca2+-sensitive K+ channels (KCa(ACh) channels). The extracellular application of gentamicin and neomycin reversibly blocked ACh-evoked K+ current (IK(ACh)) with IC50 values of 5.5 and 3.2 microM, respectively. The results showed that the blocking mechanism of IK(ACh) was due to inhibition of Ca2+ influx via nAChRs. Our study also provides interesting insights into the functional coupling between nAChRs and KCa(ACh) channels in OHCs. By directly recording the cation current flowing through nAChRs (In(ACh)) using an intracellular solution containing 10 mM BAPTA, we measured an EC50 near 110 microM for ACh-evoked In(ACh). This EC50 for ACh is one order of magnitude higher than that measured indirectly on IK(ACh). This reveals a rather low affinity of ACh for its receptor but a very efficient coupling between nAChRs and KCa(ACh) channels. We also show that a high external Ca2+ concentration reverts the gentamicin inhibition of IK(ACh) and that gentamicin directly alters the cation current flowing through the nAChRs of OHCs. We propose that gentamicin acts as a non-competitive cholinergic blocker by displacing Ca2+ from specific binding sites at the nAChRs. This block of the nAChRs at the level of the postsynaptic membrane in OHCs could explain the inhibitory effect of gentamicin reported on the crossed medial olivocochlear efferent system in vivo.
Blanchet
C
C
Laboratoire de Biologie Cellulaire et Moléculaire de l'Audition, Equipe Mixte INSERM 99-27, Université de Bordeaux 2, CHU Hôpital Pellegrin, 33076 Bordeaux, France.
Erostegui
C
C
Sugasawa
M
M
Dulon
D
D
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005839:Gentamicins / Q000494:pharmacology*; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000737:chemistry / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D011950:Receptors, Cholinergic / Q000502:physiology*; D014665:Vasodilator Agents / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D005839:Gentamicins; D015221:Potassium Channels; D011950:Receptors, Cholinergic; D014665:Vasodilator Agents; D000109:Acetylcholine; D002118:Calcium