Gentamicin blocks ACh-evoked K+ current in guinea-pig outer hair cells by impairing Ca2+ entry at the cholinergic receptor

Abstract

Aminoglycoside antibiotics such as gentamicin are known to block the medial olivocochlear efferent system. In order to determine whether this inhibition takes place at the postsynaptic cholinergic receptors in outer hair cells (OHCs), we studied the effects of these polycationic molecules on cholinergic currents evoked in isolated guinea-pig OHCs. The cholinergic response of OHCs involves nicotinic-like receptors (nAChRs) permeable to Ca2+ ions that activate nearby Ca2+-sensitive K+ channels (KCa(ACh) channels). The extracellular application of gentamicin and neomycin reversibly blocked ACh-evoked K+ current (IK(ACh)) with IC50 values of 5.5 and 3.2 microM, respectively. The results showed that the blocking mechanism of IK(ACh) was due to inhibition of Ca2+ influx via nAChRs. Our study also provides interesting insights into the functional coupling between nAChRs and KCa(ACh) channels in OHCs. By directly recording the cation current flowing through nAChRs (In(ACh)) using an intracellular solution containing 10 mM BAPTA, we measured an EC50 near 110 microM for ACh-evoked In(ACh). This EC50 for ACh is one order of magnitude higher than that measured indirectly on IK(ACh). This reveals a rather low affinity of ACh for its receptor but a very efficient coupling between nAChRs and KCa(ACh) channels. We also show that a high external Ca2+ concentration reverts the gentamicin inhibition of IK(ACh) and that gentamicin directly alters the cation current flowing through the nAChRs of OHCs. We propose that gentamicin acts as a non-competitive cholinergic blocker by displacing Ca2+ from specific binding sites at the nAChRs. This block of the nAChRs at the level of the postsynaptic membrane in OHCs could explain the inhibitory effect of gentamicin reported on the crossed medial olivocochlear efferent system in vivo.

Figure 1. I_K(ACh) depends on a Ca²⁺ influx

A, effect of lowering extracellular Ca²⁺ concentration on I_K(ACh). ACh (100 μM) was sequentially applied with 1 min intervals between applications. Stimulation was via two puff pipettes containing either a normal carrier solution (1.26 mM CaCl₂: ACh100) or a carrier solution with a tenfold lower concentration of Ca²⁺ (0.126 mM CaCl₂: ACh100Ca/10). V_h was set at -40 mV. B, effect of ACh100 and ACh100Ca/10 at different membrane potentials. The cell was stimulated by a slow protocol (left) consisting of 3 s voltage steps to between -90 and +50 mV in 20 mV increments triggered from a V_h of -60 mV. Voltage steps were spaced 30 s apart. ACh100 (middle) or ACh100Ca/10 (right) was applied (horizontal bar) for 500 ms at each voltage step, 600 ms after its onset. C, effect of varying the V_h during an ACh100 or ACh100Ca/10 application. This fast protocol (left) consisted of 80 ms voltage steps to between -90 and +50 mV in 20 mV increments from a V_h of -60 mV spaced 400 ms apart. The fast protocol was performed without ACh application (control), during a continuous 5 s application of ACh100, again in the absence of ACh application (control) and finally during a continuous 5 s application of ACh100Ca/10. The whole sequence was performed twice with a 1 min interval between applications. The pressure-puff application (horizontal bar) of ACh100 or ACh100Ca/10 started 1 s before the voltage-step protocol. The traces are net currents obtained after subtraction of control (leak) currents from those obtained during an ACh100 (middle) or ACh100Ca/10 (right) application. Current amplitudes reported in D were measured 8 ms after the onset of each voltage step as indicated by the arrows under the corresponding symbols. D, I-V relationships of the responses presented in B and C. Currents triggered by ACh100 (filled symbols) and ACh100Ca/10 (open symbols) were measured at their maximum amplitude for the slow protocol (squares) and as described in C for the fast protocol (circles). Filled and open arrows point to the maximum amplitude of the I–V relationships for ACh100- and ACh100Ca/10-evoked currents during the slow protocol, respectively. Recordings in A, B and C are from the same cell dialysed with internal solution 1. All these effects were reversible and repeated several times.

Figure 2. Aminoglycoside antibiotics reversibly block I_K(ACh) in a dose-dependent manner

A, effect of 50 μM gentamicin on I_K(ACh). Cholinergic responses were evoked by sequential pressure-puff applications of 100 μM ACh alone (ACh100) or in combination with 50 μM gentamicin (ACh100GM50) with 1 min intervals between applications. V_h was set at -40 mV. B, relative amplitude of I_K(ACh) as a function of aminoglycoside antibiotic (AGA) concentration. ACh was applied at 100 μM for 10 s alone and with increasing concentrations of gentamicin (•), neomycin (□) or streptomycin (*) by means of the U-tubing system. The amplitude of I_K(ACh) was measured at the steady state (i.e. at the end of each 10 s application). To maximize current sizes, measurements were taken at 0 mV, 20 ms after a voltage step from -60 mV, as previously described (Eróstegui et al. 1994). Current amplitudes were expressed relative to the initial control value for each cell. Data represent the mean value (±s.d.) with the number of cells tested indicated beside each symbol. Dose-inhibition curves are the best fits calculated from the empirical Hill equation Y= 1/(1 + (X/IC₅₀)^n_H) where X is the AGA concentration. For GM, IC₅₀= 5.5 μM and n_H= 0.95. For NM, IC₅₀= 3.2 μM and n_H= 0.38. For SM, IC₅₀ and n_H values were extrapolated to 282.6 μM and 0.66, respectively.

Figure 3. Block of I_K(ACh) by gentamicin at different membrane potentials

A, effect of 100 μM ACh alone or in combination with 50 μM GM at different membrane potentials. The cell was stimulated with a slow protocol as described in Fig. 1B. ACh100 (left) or ACh100GM50 (right) was applied for 500 ms at each voltage step, 600 ms after its onset. B, I-V relationships of the responses displayed in A, plus others obtained subsequently with a similar slow protocol but with voltage steps to between -100 and +60 mV. Currents triggered by ACh100 (▾) and ACh100GM50 (▿) were measured at their maximum amplitude (filled and open arrows, respectively). C, same experiment as in A, except that 30 μM GM, rather than 50 μM, was co-applied with ACh (ACh100GM30). D, effect of varying the V_h during an ACh100 or ACh100GM30 application using the same fast protocol as described in Fig. 1C. The traces shown are the net currents obtained after subtraction of the control (leak) currents recorded with the fast protocol in between ACh100 and ACh100GM30 applications. Current amplitudes reported in E were measured 8 ms after the onset of each voltage step as indicated by the arrows under the corresponding symbols. E, I-V relationships of the responses presented in D and C plus others as described in B. Currents triggered by ACh100 (filled symbols) and ACh100GM30 (open symbols) were measured at their maximum amplitude for the slow protocol (triangles) and as described in D for the fast protocol (diamonds). Filled and open arrows indicate the maximum amplitude of the I–V relationships for ACh100- and ACh100GM30-evoked currents during the slow protocol, respectively. A and B are from the same cell, C–E are from another cell. Both cells were dialysed with internal solution 1. All these effects were reversible and repeated several times.

Figure 4. Gentamicin impairs the Ca²⁺ influx necessary for I_K(ACh) activation

A, currents recorded using a tail protocol (bottom middle) in control conditions (top middle) or during an ACh100 (top right, filled bar) or ACh100GM30 (top left, open bar) application. The tail protocol is identical to the fast protocol described in Fig. 1C, except that voltage steps were triggered from a V_h of -10 mV; the horizontal bar indicates the timing of pressure-puff application. Net currents triggered by ACh100 (bottom right) or ACh100GM30 (bottom left) during the tail protocol were obtained after subtraction of the control leak currents recorded in between. Current amplitudes reported in B were measured 8 ms after the offset of each voltage step as indicated by the arrows under the corresponding symbols. B, I-V relationships of the tail currents presented in A. Tail current amplitudes triggered during ACh100 (♦) or ACh100GM30 (⋄) application were measured as described in A. Data presented in this figure are from the same cell as illustrated in Fig. 3C–E recorded with internal solution 1. All these effects were reversible and repeated several times.

Figure 5. Increasing the extracellular concentration of Ca²⁺ or ACh reverts the block of I_K(ACh) by gentamicin

A, example of sequential current traces evoked by 300 μM ACh alone (ACh300) or with 50 μM GM (ACh300GM50). The carrier solution was either standard (1.26 mM Ca²⁺) or supplemented with 10 mM CaCl₂ as indicated. Recording traces are separated by 1 min intervals. B, example of sequential current traces evoked by 100 μM ACh (ACh100) and by increasing concentrations of ACh (100, 300 and 500 μM) supplemented with 50 μM GM (ACh100GM50, ACh300GM50 and ACh500GM50). Recording traces are separated by 1 min intervals. A and B are from two different OHCs voltage clamped at -40 mV and dialysed with internal solution 1. Drugs were applied to both cells using the U-tubing system described in Methods, thus explaining the longer delay of the responses.

Figure 6. The dose-response relationship of the OHC cholinergic response shifts with membrane potential

A, example of cholinergic currents from two OHCs evoked by pressure-puff application of 30 μM ACh (Cell 1) or 1000 μM ACh (Cell 2) compared with 100 μM ACh (used as reference) at V_h=−65, -20 and +70 mV. Internal solution 2, containing 100 mM CsCl and 1.1 mM EGTA, was used because it allowed extended recordings at positive potentials. B, dose-response relationships of the ACh-evoked currents at three V_h values. Peak amplitudes of ACh-evoked currents were expressed relative to the corresponding reference response obtained with 100 μM ACh. Each symbol is the mean relative response from the number of cells reported vertically for each V_h. For clarity, only +s.d., –s.d. and ±s.d. are displayed (unless masked by symbols) for measurements performed at -65, -20 and +70 mV, respectively. Some cells could be successively challenged at the three V_h values as exemplified in A. For each V_h, dose-response curves were fitted with the empirical Hill equation Y= 1/(1 + (EC₅₀/X)^n_H). Best fits yield EC₅₀ values of 21, 33 and 122 μM and n_H values of 1.9, 2.3 and 1.7 at V_h=−65 mV (dashed line), -20 mV (dotted line) and +70 mV (continuous line), respectively.

Figure 7. Dose-response relationships of the I_n(ACh) at two V_h values: voltage-independent low affinity of ACh for nAChRs

A, example of cholinergic currents from four OHCs evoked by pressure-puff application of ACh at various concentrations compared with 100 μM ACh (used as reference) at V_h=−65 and +50 mV. The 10 mM BAPTA buffered internal solution 3 was used to prevent I_K(ACh) activation. The vertically aligned traces were from a single cell first challenged at -65 mV. The current traces of the four OHCs tested for increasing concentrations of ACh (as indicated in the middle) are displayed horizontally. To improve visual comparison, reference responses were scaled to the same peak amplitude both at +50 mV (top) and at -65 mV (bottom). Vertical scale bars therefore represent, from left to right traces, 167, 184, 115 and 280 pA at +50 mV and 88, 51, 70 and 143 pA at -65 mV, while horizontal scale bars represent 1 s for all traces. B, dose-response relationship of the ACh-evoked cation current at +50 mV. Peak amplitudes of ACh-evoked currents were expressed relative to the corresponding reference response at 100 μM ACh. Each symbol is the mean relative response (±s.d.) from the number of cells indicated beside. The dose-response curve was fitted with the empirical Hill equation Y= 1/(1 + (EC₅₀/X)^n_H). Best fit yields an EC₅₀ of 110 μM and a n_H of 1.8. C, the dose-response relationship of the ACh-evoked cation current at -65 mV. Plots and fits were performed as described in B but with current recorded at -65 mV. Some cells could also be challenged at +50 mV as shown in A. Data represent the mean relative response (±s.d.) with the number of cells indicated beside each symbol. Best fit yields an EC₅₀ of 114 μM and a n_H of 2.0. For clarity in B and C,±s.d. are displayed as simple bars unless they are masked by symbols.

Figure 8. Effect of gentamicin on I_n(ACh)

Sequential ACh-evoked cation currents were activated by a pressure puff of 100 μM ACh alone (ACh100) or with 50 μM GM (ACh100GM50) on a single OHC at +60 mV (top) or on another cell at -60 mV (bottom). The 10 mM BAPTA-containing internal solution 3 was used to prevent I_K(ACh) activation. Recording traces were separated by 1–2 min intervals except for the last at -60 mV, which was obtained after 20 min washout.