Effect of capsaicin on potassium conductance and electromotility of the guinea pig outer hair cell

Abstract

Capsaicin, the classic activator of TRPV-1 channels in primary sensory neurons, evokes nociception. Interestingly, auditory reception is also modulated by this chemical, possibly by direct actions on outer hair cells (OHCs). Surprisingly, we find two novel actions of capsaicin unrelated to TRPV-1 channels, which likely contribute to its auditory effects in vivo. First, capsaicin is a potent blocker of OHC K conductances (I(K) and I(K,n)). Second, capsaicin substantially alters OHC nonlinear capacitance, the signature of electromotility - a basis of cochlear amplification. These new findings of capsaicin have ramifications for our understanding of the pharmacological properties of OHC I(K), I(K,n) and electromotility and for interpretation of capsaicin pharmacological actions.

Figure 1

Capsaicin blocks I_K and I_K,n of OHCs from the upper third turn. A. representative currents (of 72 μM OHC) induced by step voltage commands following application of capsaicin (cap), 4-AP and BaCl₂. Net currents (cap, 4-AP and Ba²⁺ sensitive current) are after subtraction from control (ctrl) currents in bath. The ctrl current in the figure is for capsaicin and the ctrl for 4-AP and BaCl₂ are not shown here. The zero current is shown as a solid horizontal bar preceding the step currents. Representative I–V plots were produced for each original recordings B. representative I–V plots of net currents in A. The current for each voltage (V) is the 100 ms average bounded by the arrows. C. bar graph summary: 300 μM capsaicin, 300 μM 4-AP and 10 mM BaCl₂ blocked outward currents at 0 mV and inward currents at −100 mV. Currents were normalized by the currents in ctrl bath. * P<0.05. D. dose response curve of capsaicin sensitive current at 0 mV (n=5). Current (ratio): currents normalized by the current at 600 μM capsaicin. E. representative recording of membrane potential with 300 μM capsaicin.

Figure 2

Capsaicin blocks I_K,n of OHCs from the lower 2nd turn. A. representative currents in control (ctrl) bath and following application of 300 μM capsaicin (cap). Net currents (ctrl-cap) are after subtraction from control (ctrl) currents in bath. B. representative I–V plots of the currents in A. C. bar graph summary: 300 μM cap blocked outward currents at 0 mV and inward currents at −100 mV. Currents were normalized by the currents in ctrl. * P<0.05. D. Effect of cap (0.1 mM) on steady-state activation curve of I_K,n. Normalized tail current amplitudes versus prepulse potentials were fitted with a first-order Boltzmann function (n=6): I= I_max/(1+exp ((V−V_half)/S), where I_max is the fully activated current amplitude at the tail-current potential, V_half is the potential at half-maximal activation, V is prepulse potentials and S is the slope factors.

Figure 3

4-AP blocks the net capsaicin currents. A. representative currents in ctrl bath and following application of 300 μM capsaicin (cap). Net currents (ctrl-cap) are after subtraction from ctrl currents in bath. B. representative currents in 4-AP (300 μM) bath and following application of 300 μM cap. Net currents (cap-sensitive currents) are after subtraction from the currents in 4-AP bath. C. representative I–V curves of net currents in A and B. The current for each V is the 100 ms average bounded by the arrows.

Figure 4

BaCl₂ partially blocks the net capsaicin currents. A. representative current in gap-free mode at holding voltage of −44 mV. The vertical spike currents, such as a, b, c and d, were caused by the I–V step voltage commands as shown in B. B. net capsaicin (300 μM) currents (a–b) in control bath. The zero current was shown as a solid horizontal bar preceding the step currents in B and C. C. net capsaicin (300 OM) currents (c–d) in the bath with 10 mM BaCl₂. D. representative I–V curves of net currents in B and C. The current for each V is the 100 ms average bounded by the arrows. E. bar graph summary: the net capsaicin currents at Vh of −40 mV in the control (ctrl) and BaCl₂ (10 mM) bath. The currents were normalized by the currents of ctrl. * P<0.05, n=3.

Figure 5

Capsaicin blocks I_K and I_K,n at the concentration (20 μM) used in previous in vivo studies. Bar graph summary: 20 μM capsaicin (cap) (n=10) significantly blocked outward currents at 0 mV and inward currents at −100 mV with step protocols (shown in Fig. 1). Currents were normalized by the currents in ctrl bath. * P<0.05.

Figure 6

Representative effects of capsaicin on OHC NLC. A. Cm-Vm plots for increasing concentrations of capsaicin. Both a decrease in peak capacitance and shift in voltage at peak capacitance result from drug exposure. B. Dose response function with standard errors, showing an IC50 of 158 μM (n=8). The data are statistically significant at 10uM and above (p<0.013), showing a biphasic response shift at low concentrations. Peak NLC reduction is significant at 30 uM and above (p<0.05).

Figure 7

TRPV1 immunofluorescence labeling of guinea pig and Trpv1 ^−/− mice. A, a representative confocal projection image showing TRPV1 immunolabelling in OHCs, IHCs and pillar cells of guinea pigs. The insert shows that TRPV1 is expressed in the basolateral membrane of OHCs. No significant labeling was found in OHCs, IHCs and PCs of negative controls incuding 1) primary antibody only (B), 2) with control peptide (C) and 3) Trpv1 ^−/− mice (Fig D). TRPV1 labeling of Trpv1 ^+/+ mice is similar to guinea pigs (data not shown).