K+ channels in the inner ear : electrophysiological and molecular studies

Abstract: Potassium channels are involved in several fundamental cellular processes. Heavy metals, such as mercury and lead, are widespread toxic pollutants and have been shown to act on various ion channels in the central nervous system. In the present thesis the effects of mercury and lead compounds on potassium currents in auditory sensory hair cells were investigated. During the electrophysiological recordings a new type of ion current, an M-like potassium current, was observed. M currents, involved in e.g. stabilizing membrane excitability, have been described not only in the central nervous system but also in the peripheral visual and olfactory systems. The corresponding M channel is formed by KCNQ protein subunits. To elucidate the molecular basis of the cochlear M currents, Kcnq expression and localization of KCNQ channel proteins were pursued in the inner car. Using whole-cell patch clamp recordings it was shown that HgC12 blocked onset peak outward and inward K+ currents of guinea pig sensory outer hair cells in a voltage- and dosedependent manner, probably by modifying channel gating mechanisms. Pb2+ on the other hand did not produce any significant effects on hair cell potassium currents. Patch-clamp experiments using a particular voltage protocol revealed an M-like potassium current in guinea pig outer hair cells. The low-threshold, voltage- and timedependent, noninactivating current displayed a -75 mV reversal potential and a -35 mV halfactivation. This Mlike current was significantly reduced by cadmium chloride. Results from one-step RT-PCR indicated that genes encoding M channel subunits KCNQ2 - 5 were expressed in the mammalian cochlea. Two Kcnq2 splice variants were expressed in the rnodiolus (containing the auditory spiral ganglion neurons) and the organ of Corti. Sequence data showed that exon 15a, which is important for M current kinetics, was spliced out from both Kcnq2 transcripts. To verify the molecular substrate of the recorded M current, the presence of KCNQ channel proteins within the cochlea was investigated using immunohistochemistry. Antibodies against KCNQ2 and KCNQ3 stained both spiral ganglion cells types (type 1 and 11), and their afferents projecting to the sensory inner and outer hair cells. In the organ of Corti, hair cell bodies, nerves, nerve endings of efferent, afferent fibers stained for KCNQ2. For the sensory cells, a base-to-apex gradient was seen, while for nerve fibers and nerve endings, apex-to-base and radial gradients of KCNQ2 subunit expression were observed. KCNQ3 exhibited a similar pattern of immunostaining but without involving nerve fibers. In conclusion, although both mercury and lead are harmful to the auditory system, only Hg 2+ seems to be acting at the level of the outer hair cell K+ channels. The presence of an Mlike potassium current was shown in guinea pig outer hair cells. Its molecular substrate was further demonstrated using RT-PCR and immunohistochemistry. The functional role of M currents in the cochlea remains unclear but it is suggested that an M current may be involved in controlling hearing sensitivity by modulating outer hair cell excitability. However, more work is needed to clarify this hypothesis.