Pimozide Increases a Delayed Rectifier K+ Conductance in Chicken Embryo Vestibular Hair Cells

Abstract

Pimozide is a conventional antipsychotic drug largely used in the therapy for schizophrenia and Tourette's syndrome. Pimozide is assumed to inhibit synaptic transmission at the CNS by acting as a dopaminergic D₂ receptor antagonist. Moreover, pimozide has been shown to block voltage-gated Ca²⁺ and K⁺ channels in different cells. Despite its widespread clinical use, pimozide can cause several adverse effects, including extrapyramidal symptoms and cardiac arrhythmias. Dizziness and loss of balance are among the most common side effects of pimozide. By using the patch-clamp whole-cell technique, we investigated the effect of pimozide [3 μM] on K⁺ channels expressed by chicken embryo vestibular type-II hair cells. We found that pimozide slightly blocks a transient outward rectifying A-type K⁺ current but substantially increases a delayed outward rectifying K⁺ current. The net result was a significant hyperpolarization of type-II hair cells at rest and a strong reduction of their response to depolarizing stimuli. Our findings are consistent with an inhibitory effect of pimozide on the afferent synaptic transmission by type-II hair cells. Moreover, they provide an additional key to understanding the beneficial/collateral pharmacological effects of pimozide. The finding that pimozide can act as a K⁺ channel opener provides a new perspective for the use of this drug.

Keywords: K+ channel; balance; chicken embryo; dizziness; hair cell; ionic current; patch-clamp; pimozide; vestibular function; voltage response.

Figure 1

Representative whole-cell currents recorded from a type-II hair cell (E15). Here and in the next figures: CTRL = control condition; PMZD = pimozide; horizontal dashed line = zero-current level. (A,B), Membrane current responses recorded in control condition (A) or in the presence of pimozide [3 µM] (B) evoked by voltage steps to −40 mV, −30 mV and −20 mV after conditioning at −120 mV. The thick horizontal bar above the first region of the current indicates the holding current. The dashed rectangle indicates the time window of 17 ms considered for the expanded trace region shown in the panels at the bottom. The filled triangles refer to the peak of I_K,A.

Figure 2

Representative whole-cell currents recorded from a type-II hair cell (E15). (A,B), Membrane current responses recorded in control condition (A) or in the presence of pimozide [3 µM] (B) evoked by voltage steps as indicated next to each trace, after conditioning at −120 mV (only the last part of the response to −120 mV is shown). The dashed rectangle indicates the time window (17 ms) considered for measuring the peak current. The corresponding expanded traces are shown below each panel. The filled triangles indicate the time points at which the peak current was measured. The steady-state current was measured towards the end of the depolarizing steps (filled squares).

Figure 3

Average peak and steady-state current-voltage (I–V) curves obtained from type-II hair cells with and without pimozide (E15-E21; n = 7). Values are shown as mean ±S.E.; see Tables S2 and S3 in the Supplementary Material. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Figure 4

Normalized chord conductance/voltage relation (E15-E21; n = 7) (mean ±S.E.; see Table S4 in the Supplementary Material) for I_K,v. Fitting was performed with the Boltzmann function (see Methods). The half-activation voltage (V_1/2) was −4 mV in control and −18 mV in pimozide (see dashed lines). The slope factor was 16 in control condition and 18 in pimozide.

Figure 5

Representative whole-cell currents recorded from a type-II hair cell (E18). (A,B), Membrane current responses recorded in control condition (A) or in the presence of pimozide (B). In control condition, I_K,A was fully inactivated at the end of the 75 ms conditioning voltage of −40 mV. Note the change in the current response during the conditioning voltage of −40 mV in pimozide, consistent with the inhibition of I_K,A and the increase and acceleration of I_K,v. The filled triangles indicate the time points at which the peak current was measured. The steady-state current was measured towards the end of the depolarizing steps (filled squares).

Figure 6

I_K,v kinetics as a function of membrane voltage. (A), time-to-peak. (B), inactivation time constant. The inactivation time constant was obtained by fitting the decaying portion of the current with Equation (3) (see Methods). Not all cells showed a clear inactivation during the 1 s voltage steps less depolarized than 10 mV in control conditions, which is why values are only shown from 10 mV. Data were obtained from 8 type-II hair cells (E15-E19) following I_K,A inactivation by the 75 ms conditioning voltage of −40 mV. Values are shown as mean ±S.E.; see Tables S7 and S8 in the Supplementary Material. * p ≤ 0.05; ** p ≤ 0.01.

Figure 7

Peak and steady-state I–V relations after conditioning at −40 mV obtained from 8 type-II hair cells (E15-E19) with and without pimozide. Values are shown as mean ±S.E.; see Tables S5 and S6 in the Supplementary Material. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 8

Representative whole-cell current recorded from an E10 type-II hair cell. (A,B), Membrane current response evoked by voltage steps as indicated next to each trace, after conditioning at −120 mV, in control condition (A) or in the presence of pimozide [3 µM] (B).

Figure 9

Representative voltage responses recorded from a type-II hair cell (E20) in control condition (A) or in the presence of pimozide (B). The cell was maintained at its resting membrane potential (zero current injected) and then hyperpolarized by a negative current step of −50 pA amplitude and 150 ms duration prior to iteratively applying depolarizing current steps of 500 ms in 50 pA increments starting from +10 pA. The filled triangles indicate the time points at which the peak voltage response was measured. The steady-state voltage response was measured at the end of the depolarizing current steps (filled squares).

Figure 10

Average peak and steady-state voltage–current (V–I) relations. Values were obtained by measuring the peak and steady-state voltage response elicited by the current steps (n = 7; E15–E20), as shown by filled triangles and squares in Figure 9. Values are shown as mean ± S.E.; see Tables S10 and S11 in the Supplementary Material. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 11

Voltage-clamp experiments with pimozide 0.3 μM. (A) Representative whole-cell current recorded from an E16 type-II hair cell in control condition (top panel) or in presence of pimozide (bottom panel). Conditioning voltage: −40 mV. (B) Average peak and steady-state I–V relations after conditioning at −40 mV, obtained from 7 type-II hair cells (E15-E21), in control condition or in the presence of pimozide. (C) Average time-to-peak in control condition or in the presence of pimozide (D), average inactivation time constant in control condition or in the presence of pimozide. All values are shown as mean ±S.E. See Tables S12–S15 in the Supplementary Material. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.