Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.

Cav1.3 (alpha1D) Ca2+ currents in neonatal outer hair cells of mice.

Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.

Expression of Ca2+-activated BK channel mRNA and its splice variants in the rat cochlea.

Voltage-activated K(+) channels are important for shaping the receptor potentials of cochlear hair cells. In particular, the functional maturation of inner hair cells in mice around the onset of hearing coincides with the expression of a large, fast K(+) conductance, probably mediated by Ca(2+)-activated K(+) (BK) channels. In hearing organs of lower vertebrates, frequency tuning depends on BK-type K(+) channels with different kinetics. Kinetics are varied by alternative splicing of the channels' alpha subunits and combination with modulating beta subunits. It is unclear whether similar mechanisms "fine tune" mammalian hair cells. We used various polymerase chain reaction (PCR) approaches to screen rat cochleae for splice variants of BK-type alpha subunits. We isolated mainly minimal variants and only occasionally splice variants with additional inserts. We conclude that alpha subunits with different kinetics are not substantially used in the rat cochlea. However, we isolated six variants differing in their extreme C-terminal sequences, which may be involved in the targeting of the channel protein. By using reverse transcriptase-PCR, we demonstrated also the expression of transcripts for several beta subunits. In situ hybridization experiments revealed strict coexpression of alpha with beta1 transcripts. In inner hair cells, strong labeling emerged shortly before the onset of hearing. Labeling of outer hair cells appeared later and generally weaker. Thus, our molecular data confirm electrophysiological results that suggested that BK channels underlie the large K(+) conductance in inner hair cells of mammals. Extensive splicing of BK channel transcripts, however, does not seem to be used in mammalian hair cells as is done in lower vertebrates.