Publication Date: 2010.
In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic α9α10 receptor coupled to the activation of SK2 Ca 2+-activated K+ channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), ω-agatoxin IVA and ω-conotoxin GVIA, respectively, we show that Ca2+ entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca2+ entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca2+-activated K+ channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons. Copyright © 2010 the authors.
Author affiliation: Ballestero, J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Author affiliation: Katz, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Keywords: acetylcholine; calcium activated potassium channel; calcium channel L type; calcium channel N type; calcium channel P type; calcium channel Q type; calcium ion; calretinin; cell marker; iberiotoxin; nifedipine; nitrendipine; omega agatoxin IVA; omega conotoxin GVIA; synapsin; voltage gated calcium channel; acetylcholine release; animal cell; animal tissue; article; cochlear nerve; controlled study; Corti organ; efferent nerve; electrostimulation; female; fluorescence microscopy; hair cell; immunohistochemistry; inhibitory postsynaptic potential; isolated organ; male; mouse; nonhuman; pharmacological blocking; priority journal; protein expression; protein function; protein localization; synapse; voltage clamp; whole cell; Acetylcholine; Animals; Animals, Newborn; Biophysics; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Electric Stimulation; Female; Hair Cells, Auditory, Inner; Inhibitory Postsynaptic Potentials; Male; Mice; Mice, Inbred BALB C; Olivary Nucleus; Organ of Corti; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Synapses; Synaptic Transmission.
Repository: Biblioteca Digital (UBA-FCEN). Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales
Publication Date: 2013.
In Leydig cells, hormonal stimulation by LH/hCG entails increased intracellular Ca(2+) levels and steroid production, as well as hyperpolarization of the cell membrane. The large-conductance Ca(2+)-activated K(+)-channel (BK(Ca)) is activated by raised intracellular Ca(2+) and voltage and typically hyperpolarizes the cell membrane. Whether BK(Ca) is functionally involved in steroid production of Leydig cells is not known. In order to explore this point we first investigated the localization of BK(Ca) in human and hamster testes and then used a highly specific toxin, the BK(Ca) blocker iberiotoxin (IbTx), to experimentally dissect a role of BK(Ca). Immunohistochemistry and RT-PCR revealed that adult Leydig cells of both species are endowed with these channels. Ontogeny studies in hamsters indicated that BK(Ca) becomes strongly detectable in Leydig cells only after they acquire the ability to produce androgens. Using purified Leydig cells from adult hamsters, membrane potential changes in response to hCG were monitored. HCG hyperpolarized the cell membrane, which was prevented by the selective BK(Ca) blocker IbTx. Steroidogenic acute regulatory (StAR) mRNA expression and testosterone production were not affected by IbTx under basal conditions but markedly increased when hCG, in submaximal and maximal concentration or when db-cAMP was added to the incubation media. A blocker of K(V)4-channels, expressed by Leydig cells, namely phrixotoxin-2 (PhTx-2) was not effective. In summary, the data reveal BK(Ca) as a crucial part of the signaling cascade of LH/hCG in Leydig cells. The hyperpolarizing effect of BK(Ca) in the Leydig cell membrane appears to set in motion events limiting the production of testosterone evoked by stimulatory endocrine mechanisms.
Author affiliation: Matzkin, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina
Author affiliation: Lauf, S.. University of Munich. Anatomy III. Cell Biology; Alemania
Author affiliation: Spinnler, K.. University of Munich. Anatomy III. Cell Biology; Alemania
Author affiliation: Rossi, Soledad Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina
Author affiliation: Köhn, F.. Andrologicum; Alemania
Author affiliation: Kunz, L.. University of Munich. Department Biology II. Neurobiology; Alemania
Author affiliation: Calandra, Ricardo Saul. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina
Author affiliation: Frungieri, Monica Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina
Author affiliation: Mayerhofer, A.. University of Munich. Cell Biology; Alemania
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas