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. 2003 Dec 15;553(Pt 3):747-58.
doi: 10.1113/jphysiol.2003.053256. Epub 2003 Sep 26.

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

Marcus Michna  1 , Martina Knirsch, Jean-Charles Hoda, Stefan Muenkner, Patricia Langer, Josef Platzer, Jorg Striessnig, Jutta Engel
Affiliations

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

Marcus Michna et al. J Physiol. .

Abstract

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.

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Figures

Figure 1
Figure 1. Neonatal outer hair cells (OHCs) possess an inward current carried by Ca2+ or Ba2+
A, depolarisation-evoked peak inward current traces of an OHC (postnatal day 6 (P6), holding potential: −70 mV) with 1.3 mm Ca2+, 10 mm Ca2+ or 10 mm Ba2+ in the extracellular solution. B, corresponding peak current-voltage (I–V) curves for the different charge carriers for the cell shown in A. C, IBa (10 mm Ba2+) in another OHC (P7) was greatly diminished in the absence of permeant divalent cations as shown by the current traces recorded at −10 mV and by the corresponding peak I–V curves (D). E, average IBa density ±s.d. as a function of age. The numbers of cells for a given age are indicated in the bars.
Figure 2
Figure 2. The OHC IBa activates and deactivates rapidly
A, selected current traces evoked by depolarisations from the holding potential of −90 mV to the potentials indicated show rapid activation and deactivation of IBa in an OHC (P4). B, the I–V curve was taken at the last millisecond of the voltage pulse and fitted by a first order Boltzmann equation times the driving force, see Methods.
Figure 3
Figure 3. Inactivation behaviour of IBa and ICa in OHCs
A, pulse protocol to determine voltage-dependent inactivation. Selected voltage-activated inward current traces are shown for Ba2+ (B) and Ca2+ (C) as charge carriers. D and E, I–V curves at the beginning (closed symbols) and at the end of the conditioning pulse (open symbols) for IBa (D) and ICa (E). F, normalised inactivation curves for IBa and ICa were obtained by dividing the test pulse current (Itest) by the current amplitudes without depolarising prepulse (Icontrol), respectively. G, average normalised ICa inactivation curve as a function of membrane potential (left ordinate, Itest/Itest,max; obtained from 8 OHCs). Its slight U-shape indicates Ca2+-dependent inactivation. For comparison, the average normalised ICa peak I–V curve of the prepulse (right ordinate, Ipre/Ipre,max) is shown to illustrate the voltage of peak ICa and the subsequent decline of ICa due to the reduced driving force.
Figure 4
Figure 4. Kinetics of Ca2+-dependent inactivation during 400 ms depolarising voltage pulses
A, peak ICa traces elicited by depolarisations to +0 mV from three OHCs are shown with respective double-exponential fits (black lines). From top to bottom trace, fast time constants of inactivation were 10.4, 12.0 and 34.6 ms; slow time constants were 91.8, 130.0 and 168.0 ms, and the ratio between the amplitudes of the fast and the slow inactivation term (A1/A2, see Methods, eqn (3)) was 0.36, 0.67 and 0.36, respectively. B, average fast and slow ICa inactivation time constants ±s.d. for 8 OHCs obtained from the double-exponential fits as a function of the membrane potential.
Figure 5
Figure 5. The LTCC agonist Bay K 8644 increases the OHC IBa
A, peak current traces before (control) and during the application of 5 μm Bay K 8644 in the bath in a 4-day-old OHC. B, corresponding I–V curves taken at the last millisecond of the voltage pulse are shown.
Figure 6
Figure 6. The LTCC antagonist nifedipine partially blocks the OHC IBa
A, peak current traces before (control) and during the application of 10 μm nifedipine in the bath in an OHC (P4). B, corresponding I–V curves before and during nifedipine superfusion that were taken at the last millisecond of the voltage pulse. C, to compare I–V curves of the total current and the nifedipine-resistant current, I–V curves were scaled to their maximum. D, dose-response curve of the OHC IBa for nifedipine. For each nifedipine concentration, the percentage of IBa block was averaged for 5–10 OHCs. Fitting IBa block with a Hill equation yielded a maximum inhibition of 36.3 ± 0.9 %, an IC50 of 2.3 ± 0.1 μm and a Hill coefficient of 2.7 ± 0.4.
Figure 7
Figure 7. Detection of Cav1.3 α1 subunit transcripts by single-cell RT-PCR
RNA from IHCs, OHCs or epithelial (Hensen) cells was collected and subjected to RT-PCR as described in Methods. Three separate RNA samples obtained from pooled IHCs (6–7 cells per pool) or OHCs (21–35 cells per pool) were analysed. Lanes 1–4: control samples; lane 1: no template control; lane 2: Hepes-Hanks’ solution without cells; lane 3: OHC sample processed without reverse transcriptase; lane 4: epithelial (Hensen) cells; lanes 5–7: OHC preparations; lanes 8–10: IHC preparations.
Figure 8
Figure 8. The LTCC current is absent in OHCs from Cav1.3−/− mice
A, Ba2+ inward current traces at 0 mV in a 2-day-old Cav1.3−/− OHC in the TEA solution before and during superfusion with 5 μm Bay K 8644. B, corresponding I–V curves taken at the last millisecond of the voltage pulse show that the maximum inward control current was as small as −13 pA and was not increased by Bay K 8644. C, mean IBa densities ±s.d. for Cav1.3+/+ and Cav1.3−/− mice obtained from the number of cells indicated.
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