The Inactivating Potassium Currents of Hair Cells Isolated from the Crista Ampullaris of the Frog
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JOURNAL OF NEIJROPHYSIOLOGY Vol. 68, No. 5. November 1992. Prinred in U.S.A. The Inactivating Potassium Currents of Hair Cells Isolated From the Crista Ampullaris of the Frog C. H. NORRIS, A. J. RICCI, G. D. HOUSLEY, AND P. S. GUTH Departments of Pharmacology and Otolaryngology, Tulane University School of IWedicine, New Orleans, Louisiana 70112 SUMMARY AND CONCLUSIONS sophila (Baumann et al. 1987; Jan et al. 1977; Kamb et al. 1. A-type outward currents were studied in sensory hair cells 1987; Papazian et al. 1987). Investigations have localized isolated from the semicircular canals (SCC) of the leopard frog the sites for voltage-dependence, ion selectivity, and the ki- (Rana pipiens) with whole-cell voltage- and current-clamping netics of inactivation (Catterall 1986; Greenblatt et al. techniques. 1985; Iverson and Rudy 1988; MacKinnon et al. 1988). 2. There appear to be two classes of A-type outward-conduct- Alternate splicing of the Shaker gene has demonstrated that ing potassium channels based on steady-state, kinetic, pharmaco- multiple mRNAs can be made that produce A-channels logical parameters, and reversal potential. that differ in inactivation kinetics as well as in the recovery 3. The two classes of A-type currents differ in their steady-state from inactivation (Iverson and Rudy 1988; Timpe et al. inactivation properties as well as in the kinetics of inactivation. 1988; Zagotta et al. 1989). The steady-state inactivation properties are such that a significant portion of the fast channels are available from near the resting According to Hudspeth ( 1986), the bullfrog’s saccular potential. hair cell has seven ionic currents, including an A current 4. The inactivating channels studied do not appear to be cal- (Z*) . This transient K+ current in the saccular hair cells is cium dependent. fully inactivated at the normal resting potential (Hudspeth 5. The A-channels in hair cells appear to subserve functions and Lewis 1988)) suggesting at best a minimal role in sac- that are analogous to IA functions in neurons, that is, modulating cular hair cell function. Alternatively, Sugihara and Furu- spike latency and Q (the oscillatory damping function). The A- kawa ( 1989) have suggested that the A-currents in saccule currents appear to temporally limit the hair cell voltage response hair cells may be involved in modulating the damping func- to a current injection. tion of the membrane oscillations typical of these cells. Murrow and Fuchs ( 1990) have described an A-type con- INTRODUCTION ductance in the bird basilar papilla that was found exclu- sively in the short hair cells and although not in a physio- The transient outward K+ current, which has come to be logically active range, could be enabled during hyper- called Z*, was first described by Connor and Stevens polarization induced by Acetylcholine, the major efferent ( 197 1). They separated Z* from the delayed rectifier transmitter. current by the use of a protocol that elicited all currents, In contradistinction to the auditory hair cells, in a prelim- then only the noninactivating currents. Subtraction of these inary study of the currents of hair cells isolated from the two currents revealed the existence of the A current. The posterior semicircular canal (SCC) of the frog, Rana pi- protocol that elicited both inactivating and noninactivating piens Housley et al. ( 1989), suggested that an active Z* currents employed a hyperpolarizing prepulse to enable the could be elicited by depolarization from normal resting po- inactivating conductances and thus allow the channels to tentials. Correia et al. ( 1989) and Rennie and Ashmore open when the cell was depolarized. The second protocol ( 199 1) have described an A-type current in bird and guinea used a depolarizing prepulse, which inactivated the A-type pig semicircular canal, respectively. Although the steady- conductances, leaving only the noninactivating conduc- state properties allow these channels to be active in a more tances. physiological range, the relative proportion of the A-chan- In addition, these two potassium currents can sometimes nels is significantly less than that found in the frog. be separated pharmacologically. In some cells, ZA is less sen- A-type currents may be of fundamental importance in sitive to inhibition by tetraethylammonium (TEA) and SCC hair cells. We, therefore, made a more rigorous study more sensitive to block by 4-aminopyridine (4.AP) than of these inactivating outward currents revealing the exis- the delayed rectifier (Rudy, 1988; Thompson, 1977). Thus tence of two classes of A-type channels, differing largely in the definition of A-type channels is that they are voltage- their inactivation properties. sensitive, rapidly-activating, rapidly-inactivating, outward- flowing potassium channels that are blocked by 4-AP. METHODS Several roles have been suggested for A currents. In neu- rons I) they could control the spike latency, 2) they could Zsolation of hair cells regulate interspike interval, and 3) they could contribute to The procedure adopted essentially follows that reported by action potential repolarization (Rudy, 1988). Lewis and Hudspeth (1983), Hudspeth and Lewis (1988), and A great deal of information about A-channels has come Housley et al. ( 1989) for the isolation of hair cells from frog vestib- from the discovery of the Shaker mutant genes of the dro- ular organs. The external medium used by Lewis and Hudspeth 1642 0022-3077192 $2.00 Copyright 0 1992 The American Physiological Society INACTIVATING CURRENTS IN VESTIBULAR HAIR CELLS 1643 TABLE 1. Media decapitated.With the head bathed in external medium (Table 1) and sectionedsagittally, the inner ear wasexposed by openingthe Dissociation, mM External, mM Internal, mM otic capsule.The semicircularcanals were dissectedfree from the rest of the membranouslabyrinth and trimmed leaving only the CaCIZ . 2Hz0 0.02 2 1 ampullae. Thesewere openedand placed in dissociationmedium KC1 3 3 115 (Table 1) containing the proteolytic enzyme papain (0.1 mg/ml; MgC12 l HZ0 1 3 CalbiochemNo. 5 125) and L-cysteine(0.33 mg/ml) for 5 min. NaCl 122 119 The tissueswere then washedin dissociationmedium containing NaH,PO, 2 2 Na,HPO, 8 8 bovine serum albumin (0.5 mg/ml), and the cristaewere moved KH2P0,, 1 to a glass-bottomedbath on an inverted microscope.Mechanical K2HP04 4 twisting of the tissuesthen freed the individual hair cellsfrom the ATP 3 rest of the crista. The bathing medium was then changedfrom EGTA 11 dissociationmedium to external medium (Table 1). D-glucose 3 3 3 Studieswith whole-cell recordingsrequired the use of various 4-AP 10 solutions (Table 1). The bath (2.ml vol) containing the isolated TEA 10 hair cells was perfused with external medium at a rate of l-2 GTP 0.5 ml/min, and all experimentswere carried out at room tempera- ture (21°C). EGTA, ethylene glycol-bis(P-aminoethyl ether)-IV,N,N’,N’-tetraacetic acid; 4-AP, 4-aminopyridine; TEA, tetraethylammonium chloride; GTP, Cells were selectedfor whole-cell patch clamping accordingto guanosine triphosphate. the criteria enumeratedby Housley et al. ( 1989). ( 1983) contains Hepes buffer. In our procedure, this has been Whole-cell recording replacedby phosphatebuffer ( Table 1) (Norris and Guth 1985) . Cells were isolatedas previously described( Housley et al. 1989). Gigohm seals( 1- 15 GQ) were obtained with 1.5-mmOD boro- Leopard frogs (Rana pipiens) were chilled, pithed, and then silicateglass pipettes (Frederick Haer, capillary tubing No. 30-32- 000 pA Ii- 0 rn¶ 00 PA Intermediate L 0 ms FIG. 1. Current vs. time responses for three different cells (top to bottom) to 2 different whole-cell voltage-clamp proto- cols (A and B) . A : the cell is first voltage clamped to a conditioning prepulse of - 130 mV from a holding potential of -60 mV. After the 80 ms, - 130 mV prepulse the cell is depolarized for 200 ms to a given voltage step and then returned to the -60 mV holding potential. This cycle is repeated every 5 s with a 10 mV more positive step utilized each cycle. Depolarizing voltage steps ranged from - 130 to + 120 mV. This protocol is designed to elicit the inactivating currents as well as the noninactivating channel types such as the delayed rectifier, or the calcium-dependent potassium conductances. B: current responses to a similar protocol are depicted except that the conditioning prepulse has been changed to -20 mV. This protocol has been designed to elicit the noninactivating currents (see Fig. 5 ) . C: current responses are the differences (the inactivating currents) between those in A and those in the B. 1644 NORRIS, RICCI, HOUSLEY, AND GUTH 1) pulled to a resistanceof -3-5 Ma. A continuous single-elec- Drug solutions(4-AP, 1O-20 mM ) were made up in external trode patch-clamp amplifier ( Axopatch- 1B, Axon Instruments) medium (Table 1). The NaCl concentration was adjustedto ac- was usedto record from crista hair cellsduring voltage or current commodate the drug and maintain osmotic pressure.4-AP was clamp (after Hamill et al. 1981). Unlessspecified, records were obtained from Sigma.It wasapplied by bath substitution for 3 to 5 low-passfiltered at 5 kHz with a four-pole Besselfilter. Command min (i.e., 1.5-3~ bath volume) beforethe voltage-steppingproto- potentialsand currents werecontrolled with a 12.bit digital-to-an- col designedto elicit the inactivating current was applied to the alogconverter, and data were sampleddigitally at 2OO-psintervals cells( seeFig. 7 ) . Changesin inactivating currentscaused for exam- with a 12.bit analog-digitalconverter (Labmaster,Tekmar Indus- ple by 4-AP exposurewere expressedas percent of maximal inacti- tries) coupled to a microcomputer (PC’s Limited 80286, Dell vating current elicited in that cell. The currents were elicited once Computer). Clamp speed,which is dependenton uncompensated beforedrug application, at leasttwice during drug application, and seriesresistance and cell capacitance,was estimatedto be 15 ps.