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Gated Calcium Currents in Turtle Auditory Hair Cells

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Gated Calcium Currents in Turtle Auditory Hair Cells J Physiol (2003), 549.3, pp. 697–717 DOI: 10.1113/jphysiol.2002.037481 © The Physiological Society 2003 www.jphysiol.org Biophysical and pharmacological characterization of voltage- gated calcium currents in turtle auditory hair cells M. E. Schnee and A. J. Ricci Neuroscience Center and Kresge Hearing Laboratories, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA Hair cell calcium channels regulate membrane excitability and control synaptic transmission. The present investigations focused on determining whether calcium channels vary between hair cells of different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a different function. To this end, turtle auditory hair cells from high- (317 ± 27 Hz) and low- frequency (115 ± 6 Hz) positions were voltage clamped using the whole-cell recording technique, Journal of Physiology and calcium currents were characterized based on activation, inactivation and pharmacological properties. Pharmacological sensitivity to dihydropyridines (nimodipine, Bay K 8644), benzothiazepines (diltiazem) and acetonitrile derivatives (verapamil, D600) and the insensitivity to non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels were present. Fast activation rise times (< 0.5 ms), hyperpolarized half-activation potentials and a relative insensitivity to nimodipine suggest the channels were of the a1D (CaV1.3) variety. Although no pharmacological differences were found between calcium currents obtained from high- and low- frequency cells, low-frequency cells activated slightly faster and at hyperpolarized potentials, with half-activating voltages of _43 ± 1mV compared to _35 ± 1mV. Inactivation was observed in both high- and low-frequency cells. The time course of inactivation required three time constants for a fit. Long depolarizations could result in complete inactivation. The voltage of half-inactivation was _40 ± 2 mV for high-frequency cells and _46 ± 2 mV for low-frequency cells. Calcium channel inactivation did not significantly alter hair cell electrical resonant properties elicited from protocols where the membrane potential was hyperpolarized or depolarized prior to characterizing the resonance. A bell-shaped voltage dependence and modest sensitivities to intracellular calcium chelators and external barium ions suggest that inactivation was calcium dependent. (Received 12 December 2002; accepted after revision 4 April 2003; first published online 9 May 2003) Corresponding author A. J. Ricci: Neuroscience Center and Kresge Hearing Laboratories, 2020 Gravier Street Suite D, LSU Health Sciences Center, New Orleans, LA 70112, USA. Email: [email protected] Calcium channels are fundamental to signal processing in Synaptic transmission is driven by calcium entering hair auditory sensory hair cells, regulating both the membrane cells through calcium channels. Calcium channels are excitability and neurotransmitter release (Roberts et al. clustered, presumably at synaptic release sites (Roberts et 1990). Electrical resonance, the ability of the hair cell’s al. 1990; Issa & Hudspeth, 1994; Tucker & Fettiplace, membrane potential to oscillate at a particular frequency, 1995). The number of calcium channels and the number of is the primary tuning mechanism of auditory hair cells in release sites, but not the density of channels, increases with lower vertebrates (Crawford & Fettiplace, 1978; Ashmore, characteristic frequency (Sneary, 1988; Wu et al. 1996; 1983; Lewis & Hudspeth, 1983; Fuchs et al. 1988). Ricci et al. 2000). Whether calcium channels linked to Electrical resonance is driven by the interaction between neurotransmitter release are different from those linked to calcium channels and calcium-activated potassium (BK) electrical resonance remains to be elucidated. channels (Art et al. 1986; Hudspeth, 1986; Art & Fettiplace, Calcium channels have been classified biophysically, 1987). Tonotopic variations in the magnitude of both pharmacologically and molecularly (see Hille, 2001 for channel types as well as kinetic and calcium sensitivity review). L-type calcium channels typically activate at differences in the BK channels underlie the tonotopic depolarized potentials, are sensitive to dihydropyridines distribution of resonant properties (Art & Fettiplace, 1987; and show slow inactivation (Tsien et al. 1988). Calcium Fuchs et al. 1988; Hudspeth & Lewis, 1988b; Fuchs & channels are multimeric, containing , , 2 and Sokolowski, 1990; Art et al. 1995; Wu et al. 1995). Whether a b a d sometimes subunits. The subunits make up the pore- similar variations in kinetics or steady-state properties of g a forming region and are mandatory for channel the hair cell calcium channel occur tonotopically is unknown, functioning. L-type calcium channels have two main and is one of the questions addressed by this work. 698 M. E. Schnee and A. J. Ricci J Physiol 549.3 subtypes based on a subunits, the a1C and the a1D. The METHODS first identified and characterized L-type channel was the Tissue preparation a1C type, which is found largely in skeletal muscle and Auditory papillae were prepared as described previously heart, while the a1D is found in neuronal cells and some (Crawford & Fettiplace, 1985; Ricci & Fettiplace, 1997, 1998). epithelial cells. The a1D channels have several unusual Red-eared sliders (Trachemys scripta elegans), carapace length properties including a hyperpolarized activation curve, 8–13 cm (3–5 inches) were decapitated and the inner ear organs fast (submillisecond) activation rise times and an removed using procedures approved by the Animal Care Use insensitivity to the L-type dihydropyridine antagonists Committee at LSU Health Sciences Center and by standards established by NIH guidelines. The inner ear organs were placed (Koschak et al. 2001). Recently the a1D channels have into external solution containing (mM): 125 NaCl, 0.5 KCl, 2.8 been linked to synaptic release proteins and are thought to CaCl2, 2.2 MgCl2, 2 each of pyruvate, creatine, lactate and regulate some forms of synaptic transmission (Yang et al. ascorbate, 6 glucose and 10 Hepes. The solution was buffered to 1999). In addition, these accessory proteins can modulate pH 7.6 and had a final osmolality of 275 mosmol kg_1. The low- channel electrical properties (Yang et al. 1999). potassium solution was used to maintain the cells in the papilla in a hyperpolarized state, thus lengthening the viability of the tissue. Journal of Physiology The a1D channel type has been identified in the chick The tissue was pinned to the bottom of a Sylgard-coated dish with auditory papilla (Kollmar et al. 1997a, b), frog saccule minutien pins with the auditory papilla facing upward. The (Rodriguez-Contreras & Yamoah, 2001), trout saccule external membrane was removed, exposing the tectorial and mammalian cochlea (Zhang et al. 1999; Platzer et al. membrane. Protease type XXIV (Sigma) 0.02–0.04 mg ml_1 was 2000; Koschak et al. 2001). Hair cell calcium channels are added to the solution and the tissue was incubated for 5–20 min, somewhat different from the 1D channels that are depending on enzyme potency. The tectorial membrane was a removed and the enzyme washed out with multiple rinses of expressed heterologously in vitro, in particular with regard external solution. The papilla was trimmed and placed into a to inactivation. Another purpose of the present work was recording chamber with a coverslip at its base. The tissue was held to compare the properties of hair cell calcium channels to in place with three single strands of dental floss. The recording those reported for expressed a1D channels. chamber was perfused at a rate of 0.5–1 ml min_1 with external solution supplemented with 100 nM apamin (Calbiochem) to Several different types of calcium channel have been block the caesium-permeable SK calcium-activated potassium identified in hair cells. L-type channels have been current (for example see Fig. 1B; Tucker & Fettiplace, 1996). A identified in a variety of hair cell organs including the frog peristaltic pump (Gilson) was used for the bath perfusion. In saccule (Hudspeth & Lewis, 1988a; Roberts et al. 1990), recordings from low-frequency cells, 100 mM 4-aminopyridine turtle papilla (Art et al. 1986; Art & Fettiplace, 1987), chick (4-AP) was included to block any delayed-rectifier conductance papilla (Fuchs et al. 1990; Zidanic & Fuchs, 1995; Spassova (Goodman & Art, 1996). et al. 2001), guinea-pig cochlear hair cells (Bobbin et al. Recording procedures 1990; Nakagawa et al. 1991; Oshima et al. 1996; Zhang et al. A large blunt pipette was advanced into the papilla from the 1999) and the frog semicircular canal (Prigioni et al. 1992; abneural edge while applying pressure to the back end of the pipette, making a hole from which 1–3 cells could be removed to Martini 2000). N-, R- and T-type channels have also et al. ensure good access (Fig. 1A). The space around the cell being been described in vestibular hair cells (Rennie & Ashmore, recorded was carefully cleared before the cell was patched. Whole- 1991; Martini et al. 2000; Rispoli et al. 2000). In particular, cell recordings were obtained as has been described previously N-type channels have been identified recently in frog (Ricci & Fettiplace, 1997). An EPC8 (Heka) or an Axopatch 1D saccule hair cells, an end-organ traditionally thought to (Axon Instruments) was used for all recordings. The EPC8 was have only L-type channels (Su et al. 1995; Rodriguez- used for current-clamp
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