Frog saccular hair cells dissociated with protease VIII exhibit inactivating BK currents, K(V) currents, and low-frequency electrical resonance.
Outward K currents and electrical resonance of frog (Rana esculenta) saccular hair cells isolated enzymatically with bacterial protease VIII were investigated using the perforated patch-clamp method. Under voltage-clamp conditions we identified two K currents, a voltage-dependent K (K(V)) current, and a partially inactivating iberiotoxin-sensitive K (BK) current. The K(V) current activated at a membrane potential of approximately -50 mV (from a holding potential of -70 mV). Its activation rate was rather slow, having a time constant in the range 5-8 ms at 0 mV. The K(V) current was resistant to tetraethylammonium (10 mM), but was inhibited by 4-aminopyridine (1 mM). A striking feature of the BK current was its inactivation; this was monoexponential and had fast kinetics (tau(inact)=2.7 ms +/-1.2, at -10 mV; n=8). Inactivation of the current was incomplete, a residual sustained component remaining. This varied considerably among hair cells (mean ratio between peak transient and sustained component was 1.22+/-0.18, range 0.53-1.8; n=8). In current-clamp mode steady depolarizing current pulses evoked membrane potential oscillatory responses, with mean frequencies varying between 30 and 100 Hz for membrane potentials from -60 to -40 mV (n=18). Most hair cells (14/18) exhibited damped oscillations, and in the remainder a few initial damped oscillations were succeeded by smaller, undamped voltage oscillations. The peak quality factor and the characteristic frequency assessed on 14 cells displaying only damped oscillatory responses were 2.4+/-1.3 and 59+/-39 Hz, respectively. In contrast, papain-dissociated frog saccular hair cells possess solely a sustained BK current, and exhibited significantly higher resonant frequencies and quality factors. In conclusion, the K currents and the electrical resonance of hair cells dissociated in protease VIII differ markedly from those dissociated with papain, but are similar to those reported for in situ preparations, suggesting that our dissociation procedure preserves the electrophysiological profile of in situ frog saccular hair cells.
Catacuzzeno
Luigi
L
Dipartimento di Biologia Cellulare e Molecolare, Universita' di Perugia, via Pascoli 1, I-06100 Perugia, Italy.
Fioretti
Bernard
B
Perin
Paola
P
Franciolini
Fabio
F
D000818:Animals; D004553:Electric Conductivity; D010450:Endopeptidases / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000378:metabolism*; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials; D009991:Oscillometry; D010206:Papain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology*; D011893:Rana esculenta; D012444:Saccule and Utricle / Q000294:innervation*
D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D024642:Potassium Channels, Voltage-Gated; D010450:Endopeptidases; D010206:Papain