Korean J Physiol Pharmacol.  2013 Dec;17(6):537-546. 10.4196/kjpp.2013.17.6.537.

Potassium Currents in Isolated Deiters' Cells of Guinea Pig

• Chung JW
• Nam EC
• Kim WT
• Youm JB
• Leem CH

• KMID: 2285482
• DOI: http://doi.org/10.4196/kjpp.2013.17.6.537

Abstract

Deiters' cells are the supporting cells in organ of Corti and are suggested to play an important role in biochemical and mechanical modulation of outer hair cells. We successfully isolated functionally different K+ currents from Deiters' cells of guinea pig using whole cell patch clamp technique. With high K+ pipette solution, depolarizing step pulses activated strongly outward rectifying currents which were dose-dependently blocked by clofilium, a class III anti-arrhythmic K+ channel blocker. The remaining outward current was transient in time course whereas the clofilium-sensitive outward current showed slow inactivation and delayed rectification. Addition of 5 mM tetraethylammonium (TEA) further blocked the remaining current leaving a very fast inactivating transient outward current. Therefore, at least three different types of K+ current were identified in Deiters' cells, such as fast activating and fast inactivating current, fast activating slow inactivating current, and very fast inactivating transient outward current. Physiological role of them needs to be established.

Keyword

Dieters' cell; Hearing; Inner ear; Organ of Corti; Pharmacology; Potassium channels

MeSH Terms

Animals
Ear, Inner
Guinea Pigs*
Guinea*
Hair
Hearing
Organ of Corti
Pharmacology
Potassium Channels
Potassium*
Quaternary Ammonium Compounds
Tetraethylammonium
Potassium
Potassium Channels
Quaternary Ammonium Compounds
Tetraethylammonium

Figure

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  Fig. 1 Isolated Deiters' cell. The cell is easily recognized by its long phalangeal process (arrow) and nucleus in cell body.

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  Fig. 2 Activated current traces (A) and I-V curve (B) recorded from Deiters' cells. This shows rapid activating outward rectifying current. Holding at -40 mV in whole cell recording, step pulse with 10 mV increment from -110 mV to 50 mV elicited outward current, showing very strong outward rectification.

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  Fig. 3 Effects of extracellular calcium change on the activated current traces and their I-V curves. (A) 2 mM Ca2+, (B) 0 mM Ca2+, (C) I-V curves. 2 mM Ca2+ (●) and 0 mM Ca2+ (■).

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  Fig. 4 Voltage dependent changes of the reactivation time courses. (A) The voltage of prepulse was -80 mV. (B) The voltage of prepulse was -40 mV. (C) The time constant of the current reactivation was 19 msec. (D) The time constant of the current reactivation was 512 msec.

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  Fig. 5 Steady state inactivation and activation curves. (A) Current traces of the steady state inactivation. (B) Current traces of the activation. (C) The activation curve is fitted by double Boltzmann equation suggesting the presence of more than two channels. Vh: half activation (or inactivation) voltage, K: slope factor. Inactivation curve could be fitted by single Boltzmann equation and the half inactivation voltage (Vh) was -29.72 mV and the slope factor (s) was 7.36. Activation curve was fitted by double Boltzmann equation. The half activation voltages (Vh) and slope factors (s) were -10.12 mV and 4.28 for the first one and 8.21 mV and 9.73 for the second one, respectively.

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  Fig. 6 Dose dependent effect of clofilium and TEA. (A) Current traces with different concentration of clofilium. (B) Current traces with different concentration of TEA. (C) Dose response curves. Effect of TEA on the peak current amplitude (●) and on the current amplitude at 100 msec (◯) (1 mM, n=4; 5 mM, n=11; 40 mM, n=7; 149.8 mM, n=3). Effect of clofilium on the peak current amplitude (▼) and on the current amplitude at 100 msec (△) (3 µM, n=5; 30 µM, n=12; 50 µM, n=5; 100 µM, n=5).

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  Fig. 7 Isolation of three different types of currents in Deiters' cells. At a concentration of 30 µM of clofilium, control outward rectifying current (A) was blocked leaving transient outward current (B). This residual current was further blocked by 5 mM TEA and only a small transient outward current was identified (C). Clofilium-sensitive current (D) was obtained by subtracting (B) from (A). TEA-sensitive current (E) was obtained by subtracting (C) from (B).

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  Fig. 8 Voltage-current relationship of three currents. (A) The I-V curves of three currents were similar to that of control currents. The peak current amplitude was used. All the curves showed outward rectification. ●: control current (n=12); ◯: clofiliumsensitive current (n=9); ▼: TEA-sensitive current (n=9); △: clofilium and TEA resistant current (n=9). (B) The time constant (tau) was 505±160 ms in the clofilium-sensitive current, 27.6±3.1 ms in the TEA-sensitive current and 5.6±0.1 ms in the residual current (n=9).

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  Fig. 9 Characteristics of activation and inactivation of three different currents. the clofilium-sensitive current (●); TEA-sensitive current (◯); clofilium and TEA resistant current (▼). All the currents were fitted by each single Boltzmann equation. Each fitting parameters were shown on the figure.

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  Fig. 10 Validation of our mathematical model. Reactivation time courses from prepulse at -80 mV (A) and -40 mV (B) were obtained by our mathematical simulation. By plotting current density (pA/pF) against length of prepulse and fitting them to single exponential functions (C, D), time constants of reactivation were estimated to be 21 msec at -80 mV and 647 msec at -40 mV, respectively which is comparable to experimentally obtained values in Fig. 4 (19 msec at -80 mV and 512 msec at -40 mV).

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  Fig. 11 Mathematical simulation of outward K+ current and clofilium effect in Deiters' cells. Fast activating and fast inactivating current (B) and fast activating and slow inactivating current (C) were summated to reproduce experimentally obtained outward rectifying K+ current in Fig. 7A. Summated current (A) clearly shows biphasic time course of inactivation. The pulse protocol is identical with that used in Fig. 7A. Incorporation of transition from open to blocked state into the C-O-I model successfully reproduced the effect of clofilium in Fig. 7B. Time course and voltage-dependence of simulated current (D) are similar with those of experimentally obtained current in Fig. 7B. Simulated clofilium-sensitive current (E) was obtained by subtracting (D) from (A). Time course and voltage-dependence of simulated current are very similar to those of experimentally obtained current in Fig. 7D.

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