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Functional interactions between ligand-gated, voltage-, and Ca(2+)-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca(2+)-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea alpha9 and alpha10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca(2+)-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus alpha9/alpha10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the alpha9/alpha10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response. [ABSTRACT FROM AUTHOR]

The molecular identity of non-Cav1.3 channels in auditory and vestibular hair cells has remained obscure, yet the evidence in support of their roles to promote diverse Ca2+-dependent functions is indisputable. Recently, a transient Cav3.1 current that serves as a functional signature for the development and regeneration of hair cells has been identified in the chicken basilar papilla. The Cav3.1 current promotes spontaneous activity of the developing hair cell, which may be essential for synapse formation. Here, we have isolated and sequenced the full-length complementary DNA of a distinct isoform of Cav3.1 in the mouse inner ear. The channel is derived from alternative splicing of exon14, exon25A, exon34, and exon35. Functional expression of the channel in Xenopus oocytes yielded Ca2+ currents, which have a permeation phenotype consistent with T-type channels. However, unlike most multiion channels, the T-type channel does not exhibit the anomalous mole fraction effect, possibly reflecting comparable permeation properties of divalent cations. The Cav3.1 channel was expressed in sensory and nonsensory epithelia of the inner ear. Moreover, there are profound changes in the expression levels during development. The differential expression of the channel during development and the pharmacology of the inner ear Cav3.1 channel may have contributed to the difficulties associated with identification of the non-Cav1.3 currents.

The simplest cellular model for classical conditioning in the nudibranch mollusk, Hermissenda crassicornis, involves the presynaptic hair cells and postsynaptic photoreceptors. Whereas the cellular mechanisms for postsynaptic photoreceptors have been studied extensively, the presynaptic mechanisms remain uncertain. Here, we determined the phenotype of the voltage-dependent Ca(2+) current in the presynaptic hair cells that may be directly involved in changes in synaptic efficacy during classical conditioning. The Ca(2+) current can be classified as a P-type current because its activation voltage under seawater recording conditions is approximately -30 mV, it showed slow inactivation, and it is reversibly blocked by omega-agatoxin-IVA. The steady-state activation and inactivation curves revealed a window current, and the single-channel conductance is approximately 20 pS. The P-type current was enhanced by cAMP analogs (approximately 1.3-fold), and by forskolin, an activator of adenylyl cyclase (approximately 1.25-fold). In addition, the P-type current showed voltage-dependent facilitation, which is mediated by protein kinase A (PKA). Specifically, the PKA inhibitor peptide [PKI(6-22)amide] blocked the enhancement of the Ca(2+) current produced by conditioning depolarization prepulses. Because neurotransmitter release is mediated by Ca(2+) influx via voltage-gated Ca(2+) channels, and because of the nonlinear relationship between the Ca(2+) influx and neurotransmitter release, we propose that voltage-dependent facilitation of the P-type current in hair cells would produce a robust change in synaptic efficacy.

We assessed the functional determinants of the properties of L-type Ca(2+) currents in hair cells by co-expressing the pore-forming Ca(V)1.3alpha(1) subunit with the auxiliary subunits beta(1A) and/or alpha(2delta). Because Ca(2+) channels in hair cells are poised to interact with synaptic proteins, we also co-expressed the Ca(V)1.3alpha(1) subunit with syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome associated protein of 25 kDa (SNAP25). Expression of the Ca(V)1.3alpha(1) subunit in human embryonic kidney cells (HEK 293) produced a dihydropyridine (DHP)-sensitive Ca(2+) current (peak current density -2.0 +/- 0.2 pA/pF; n = 11). Co-expression with beta(1A) and alpha(2delta) subunits enhanced the magnitude of the current (peak current density: Ca(V)1.3alpha(1) + beta(1A) = -4.3 +/- 0.8 pA/pF, n = 10; Ca(V)1.3alpha(1) + beta(1A) + alpha(2delta) = -4.1 +/- 0.6 pA/pF, n = 9) and produced a leftward shift of approximately 9 mV in the voltage-dependent activation of the currents. Furthermore, co-expression of Ca(V)1.3alpha(1) with syntaxin/VAMP/SNAP resulted in at least a twofold increase in the peak current density (-4.7 +/- 0.2 pA/pF; n = 11) and reduced the extent of inactivation of the Ca(2+) currents. Botulinum toxin, an inhibitor of syntaxin, accelerated the inactivation profile of Ca(2+) currents in hair cells. Immunocytochemical data also indicated that the Ca(2+) channels and syntaxin are co-localized in hair cells, suggesting there is functional interaction of the Ca(V)1.3alpha(1) with auxiliary subunits and synaptic proteins, that may contribute to the distinct properties of the DHP-sensitive channels in hair cells.