INTRODUCTION

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Voltage-sensitive ionic currents flowing across the basolateral surfaces of hair cells can shape receptor potentials and thereby modify neurotransmitter release and the response of afferent neurons. The importance of basolateral currents is clear in auditory and vibratory organs of lower vertebrates, where such currents participate in the electrical tuning of hair cells (Fettiplace and Fuchs 1999). Basolateral currents have also been described in vestibular organs, which monitor head movements (Correia et al. 1989; Masetto and Correia 1997; Masetto et al. 1994, 2000; Ohmori 1984; Rennie and Correia 1994; Rüsch and Eatock 1996). Despite the large number of such studies, the roles of basolateral currents in vestibular processing remain a matter of speculation. In particular, it is unclear how the currents influence afferent discharge properties, which vary between different regions of the neuroepithelium (Baird and Lewis 1986; Baird et al. 1988; Boyle et al. 1991; Goldberg et al. 1990; Honrubia et al. 1989; Myers and Lewis 1990).

There may be several reasons for this situation. First, only a few studies have compared the electrophysiological properties of vestibular hair cells with their neuroepithelial locations. Such studies have been done in frog (Baird 1994a,b; Marcotti et al. 1999a,b; Masetto et al. 1994; Prigioni et al. 1996) and in bird vestibular organs (Masetto and Correia 1997; Masetto et al. 2000; Weng and Correia 1999). Regional studies of afferent discharge have been done in the frog (Baird and Lewis 1986; Honrubia et al. 1989; Myers and Lewis 1990) but not in birds. Second, the protocols used on vestibular hair cells have been of relatively short duration and have not included a background current. In these ways, the protocols may not simulate normal conditions of vestibular transduction (Goldberg and Brichta 2002). Third, to the extent that they display resonant behavior, vestibular hair cells show low-quality tuning with best frequencies of 30-100 Hz (Correia et al. 1989; Holt et al. 1999; Housley et al. 1989; Rennie and Ashmore 1991; Ricci and Correia 1999), well above the bandwidth of naturally occurring head movements (Grossman et al. 1988; Pozzo et al. 1990). A theoretical framework is needed to explain the poor and seemingly inappropriate tuning. A framework is provided in the companion paper (Goldberg and Brichta 2002), which considers how basolateral currents shape receptor potentials. A fourth reason relates to the presence of type I and II hair cells in vestibular organs.

As was first described by Wersäll (1956), type II hair cells, which are found in the vestibular organs of all vertebrates, resemble hair cells in nonvestibular organs in being innervated by bouton endings derived from several afferent and efferent fibers. Type I hair cells, which are only found in the vestibular organs of reptiles, birds, and mammals (Lewis et al. 1985; Lysakowski 1996; Wersäll and Bagger-Sjöbäck 1974), have a distinctive shape, and each of them is innervated by a calyx ending derived from a single afferent fiber. Another distinguishing feature of type I hair cells is an outwardly rectifying potassium current called I_KI (Rennie and Correia 1994) to reflect its presence in type I hair cells or I_K,L (Rüsch and Eatock 1996) because it activates at more hyperpolarized (lower, L) potentials than the outward currents of type II cells. I_K,L differs from type II currents not only in its activation range but in having slower kinetics and larger whole cell currents. Possibly distinctive roles of type I and II hair cells in vestibular transduction have been considered (Eatock et al. 1998; Goldberg 1996; Rennie et al. 1996), but none of the suggestions have been conclusively established.

The turtle posterior crista provides an opportunity to compare the respective roles of type I and II hair cells in vestibular transduction and to explore the relation between hair-cell and afferent physiology. As illustrated in Fig. 1A, the turtle posterior crista consists of two triangularly shaped hemicristae, each of which extends from the planum semilunatum to a nonsensory torus. Within a hemicrista, there is a central zone and a surrounding peripheral zone. Type I hair cells are confined to the central zone, which also contains a smaller number of type II hair cells (Brichta and Peterson 1994; Jørgensen 1974; Lysakowski 1996). Only type II hair cells are found in the peripheral zone.

View larger version (114K): [in this window] [in a new window]   Fig. 1. A: scanning electron micrograph of the turtle posterior crista as viewed from above. The crista consists of 2 hemicristae, each with a broad base abutting the planum semilunatum and a narrow apex reaching the nonsensory torus in the middle of the organ. A hemicrista has a central zone surrounded by a peripheral zone (shading). The central zone contains type I and II hair cells. Only type II hair cells are found in the peripheral zone. The areas from which hair cells were harvested are indicated on the right by boxes: torus (T), central (C), and planum (P). Note that planum harvests were confined to the corners of the neuroepithelium to minimize contamination from the central zone. Bar, 100 µm. B: photomicrographs of solitary hair cells harvested from central zone of the turtle posterior crista. Morphological scores are 1.1 (B1), 1.5 (B2), and 1.8 (B3). Bar, 10 µm. C: semithin (2-µm) sections through the middle of a hemicrista stained with azure II-methylene blue. C1 shows 2 type I hair cells enclosed by a complex calyx ending; the left-hand cell has a constricted neck but the right-hand cell does not. A type II cell is seen to the left of the complex calyx. C2, a type I cell has a neck with only a slight constriction. C3, this type I cell has a constricted neck. Bar, 10 µm.

To consider possible relations between hair-cell and afferent physiology, we briefly consider regional variations in afferent discharge properties. Vestibular-nerve afferents are referred to by the endings they possess (Fernández et al. 1988; Schessel 1982). Calyx fibers contact type I hair cells, bouton fibers terminate on type II hair cells, and dimorphic fibers contain both calyx and bouton endings and synapse on both kinds of hair cells. The central zone in the turtle posterior crista is supplied by calyx, dimorphic, and bouton fibers, while the peripheral zone is innervated only by bouton fibers (Brichta and Peterson 1994). Morphophysiological studies have related the discharge properties of afferents with the kinds and locations of the hair cells they innervate (Brichta and Goldberg 2000a,b). In their responses to head rotations, bouton afferents show a single longitudinal gradient with those ending near the planum having a more regular discharge, lower gains, and more tonic response dynamics than those ending near the torus. Calyx-bearing units, including calyx and dimorphic fibers, have an irregular discharge and can be distinguished from irregularly discharging bouton fibers by their lower gains and less phasic response dynamics.

In the present study, we used voltage-clamp protocols to characterize the voltage-sensitive ionic conductances of solitary hair cells selectively harvested from the central zone and from the peripheral zone near the planum or near the torus. Morphological criteria were used to distinguish between type I and II hair cells. We had two aims. First, we were interested in comparing the electrophysiology of the two kinds of hair cells as well as determining whether there were differences in the ionic conductances of type II hair cells from the various regions of the hemicrista. It was hoped that such information would allow us to relate afferent diversity with the electrophysiology of the corresponding hair cells. Second, we wished to characterize I_K,L in terms of its activation range, kinetics, and pharmacology. Such a characterization was of interest because of the potential importance of this conductance in determining the distinctive functions of type I hair cells and also because it provides a context for studies of synaptic transmission (Xue et al. 2000). Our investigation of I_K,L addressed two questions: 1) Did the conductance seen in the turtle resemble that described in other species? 2) Were conductances other than I_K,L present in type I hair cells? In a second paper (Goldberg and Brichta 2002), current steps and sinusoids were used to examine how the various conductances might help to determine the gain and response dynamics of vestibular transduction.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Dissociation of hair cells

Red-eared turtles (Trachemys scripta elegans, 150-250 g, 10- to 13-cm carapace length) were decapitated, their heads bisected, and the half-heads placed in a standard external solution (see Solutions). Animals were handled according to procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Chicago. The posterior ampulla was opened to reveal its crista. To loosen hair cells for mechanical dissociation, we treated the crista with the following agents dissolved in dissociation solution (see Solutions): protease XXVII (50 µg/ml, Sigma, St. Louis, MO) for 20 min; papain (500 µg/ml, Sigma) and L-cysteine (300 µg/ml, Sigma) for 45 min; and bovine serum albumin (500 µl/ml, Sigma) for 20 min. The crista was then transferred to a low-Ca²⁺ medium in a recording chamber and viewed at ×60 (Zeiss Stemi 2000 stereomicroscope). The neuroepithelium was stroked with an eyelash, releasing hair cells from one of three selected regions (peripheral zone near the planum, peripheral zone near the torus, or central zone; Fig. 1A). Given the topography of the hemicrista, it was relatively easy to get uncontaminated samples from the torus or the central zone. Our planum samples were restricted to the corners of the neuroepithelium. Even so, they could easily have been contaminated from the central zone.

Isolated cells, which were allowed to settle on the clean glass floor of the recording chamber, were viewed at ×600 with Nomarski optics on an inverted microscope (Zeiss Axiovert 100), and were continuously perfused at a rate of 500 µl/min with the standard external solution. All procedures, including recording, were done at 22°C.

Solutions

The standard external solution used for dissection and recording was a modified Leibowitz-15 medium (L-15; Gibco BRL, Buffalo, NY). Ion concentrations (in mM) were: 118 Na⁺, 4 K⁺, 4 Ca²⁺, 1 Mg²⁺, 131.5 Cl, 0.5 H₂PO₄, 5 glucose, and 5 N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES). The final osmolarity was 270 mmol/kg, and the pH of all solutions was 7.4.

Patch-clamp recording pipettes were filled with a standard internal solution containing (in mM) 140 K⁺, 0.1 Ca²⁺, 140 Cl, 2 MgATP, 10 HEPES, and 11 ethylene glycol bis-(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) with an osmolality of 260 mmol/kg and a pH of 7.4.

The dissociation solution was identical to the standard external solution except that Ca²⁺ was lowered to 100 µM by adding 1.2 mM EGTA.

When external blockers were studied, we used a phosphate-free external solution to avoid precipitates with divalent cations. The phosphate-free external solution contained (in mM) 127 Na⁺, 4 K⁺, 4 Ca²⁺, 1 Mg²⁺, 141 Cl^-, 5 HEPES, and 5 D-glucose, supplemented with 7.5 ml/l MEM vitamin mixture (Gibco BRL) and 45 ml/l MEM amino acids solution (Gibco BRL). Low concentrations of Cd²⁺ (<200 µM) and of 4-aminopyridine (4-AP; Sigma; 0.01-1 mM) were obtained by adding the blockers to the phosphate-free external solution. For experiments with external Ba²⁺ and higher concentrations of external Ca²⁺, Na⁺ concentration was reduced to preserve osmolarity. For experiments with external Cd²⁺ at concentrations exceeding 1 mM, Cd²⁺ was substituted for Ca²⁺. When external K⁺ was elevated, an equivalent amount of Na⁺ was removed.

Recording

Borosilicate pipettes were drawn and heat-polished. When filled with the standard internal solution, the pipettes had impedances of 2.5-4 M. Recordings were made in whole cell mode with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). Experiments were controlled by a Digidata 1200 interface connected to a 486-DX2 computer running pClamp 6.1 software (Axon Instruments).

A cell was first photographed with an MC 80 camera (Zeiss). Once a gigohm seal was established, the recording pipette was raised to lift the cell off the chamber floor. During these procedures, the amplifier was kept in "tracking mode," which allowed us to determine the resting or zero-current (V_Z) potential on breakthrough. We then went into voltage-clamp mode at a standard holding potential, V_H = 67 mV, chosen because it was near the mean V_Z of our cells. A standard voltage-clamp protocol was run. It consisted of eighteen 200-ms steps from V_H to voltages ranging from 137 to +33 mV in 10-mV increments. Each step was followed by a 20-ms step to 52 mV and then by a return to V_H for a duration 780 ms. All voltages have been corrected for a junction potential (see following text).

To allow for off-line calculation of series resistance (R_S) and membrane capacitance (C_M), we next recorded the currents evoked by 3-ms voltage-clamp steps of ±10 mV from a holding potential of 77 mV through a 4-pole Bessel filter with a corner frequency, f_c = 10 kHz and a sampling frequency of 100 kHz. In the analysis, the effects of the filter were removed by deconvolution. For many of our cells, a steady-state current was not abolished even when the membrane was hyperpolarized. In calculating R_S and C_M, we used equations that allowed for this possibility (Gillis 1995).

Capacitative transients were canceled, and series resistance, typically 5-15 M, was compensated by 70-90%. Compensation was calculated off-line by comparing the dial settings on the amplifier with the actual value of R_S determined from the 3-ms voltage clamps. In every cell, once compensation was achieved, the standard protocol was run once again followed by other protocols as required. Amplifier outputs were passed through 4-pole Bessel filters with a corner frequency, unless otherwise stated, of f_c = 3 kHz. Most data were sampled at 2 kHz, below the Nyquist sampling frequency of 6 kHz. Not conforming to the Nyquist theorem led to no serious problems because all of the analyses were done in the time domain and activation time constants were relatively long compared with the sampling interval.

Local exchange was used to study the effects of changing external solutions. Four glass delivery pipettes (each 200-µm diameter) were placed in parallel on a micromanipulator and were connected to separate perfusion lines. The pipettes were lowered into the bath. To deliver a particular solution, the cell was positioned within 10-20 µm of the orifice of the appropriate pipette. A peristaltic pump (Rainin) regulated fluid flow at 5 µl/min. Closure of a solenoid valve (General Valves) diverted the solution from a recirculating line to the delivery pipette. A separate valve controlled flow to each pipette. To aid in visualizing flow, we added an aqueous solution of polystyrene beads (LB-8, Sigma) to each solution at a dilution of 1:10⁶.

Morphological classification

Photomicrographs of recorded cells were projected at ×5,000 total magnification and independently ranked by two investigators without reference to the physiological results. A scale of 1.1-1.9 was used with 1.1 representing a clear type I hair cell with a constricted neck and 1.9 representing a definite type II cell with a cylindrical cell body and parallel sides. The two scores, which seldom differed by >0.1, were averaged. In the text, cells with a score of 1.1-1.3 are designated type I and those with a 1.7-1.9 score are considered type II. When the score was 1.4-1.6, the cell was placed in an "unassigned" category.

Analysis

Voltages were corrected off-line for a positive junction potential, as calculated with JPCalc software (Barry 1994), of 7 mV and for the voltage drops resulting from the incomplete compensation of series resistance.

Data were analyzed with programs written in Igor Pro software (WaveMetrics, Lake Oswego, OR), which uses the Marquardt-Levenberg nonlinear least-squares fitting algorithm. Results are expressed as means ± SE unless otherwise stated. Results were tabulated in Microsoft Excel spreadsheets. Statistical tests were run using the spreadsheet statistical functions. Probabilities listed in legends to Tables 2-4 are based on analyses of variance or covariance run in SYSTAT for the Macintosh statistical package (SYSTAT, Evanston, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This section is divided into four parts: 1) the morphological basis for assigning hair cells to type I and II categories; 2) the electrophysiology of type I and type II hair cells; 3) the biophysical properties of I_K,L, the major outwardly rectifying current of type I hair cells; and 4) the pharmacology of I_K,L.

Morphology of solitary hair cells

The only morphological feature reliably distinguishing hair cells as type I or II was the presence in the former cells of a constricted neck. Using this criterion, cells were assigned scores ranging from 1.1 to 1.9. Cells with scores of 1.1-1.3 had obviously constricted necks and were classified as type I (Fig. 1B1). Cylindrically shaped cells were scored between 1.7 and 1.9 and classified as type II (Fig. 1B3). Many cells only had slightly constricted necks and were placed in an unassigned category (score: 1.4-1.6, Fig. 1B2). A second group of unassigned cells had a round or pear-shaped appearance (not shown).

To validate our classification scheme, we applied it to semithin sections of a crista stained with azure II-methylene blue (Richardson et al. 1960). We could definitively identify hair cells in situ as type I or II by the presence or absence of a calyx ending. In semithin sections, all cells whose constricted necks would have given them scores 1.3 were of the type I variety (Fig. 1C1, left type I cell, and Fig. 1C3). On the other hand, there were some type I cells with only a slight constriction (Fig. 1C2) or none at all (Fig. 1C1, right type I cell).

Fifty type I hair cells were surveyed in semithin sections from the same crista. Of these, two would have been considered type II and five would have been left unassigned. Many type II hair cells in the same material could be followed throughout much of their length. A substantial fraction of them had a slight constriction and would have remained unassigned. None would have been assigned to the type I category.

Electrophysiology of type I and II hair cells

As was previously reported in other preparations (Correia and Lang 1990; Griguer et al. 1993a,b; Holt et al. 1999; Lennan et al. 1999; Masetto et al. 2000; Rennie and Correia 1994; Rüsch and Eatock 1996; Rüsch et al. 1998), type I and II hair cells differ in their electrophysiology. There are differences in both outwardly and inwardly rectifying currents, which we consider in turn.

OUTWARDLY RECTIFYING CURRENTS. Most type I hair cells have an outwardly rectifying current called I_KI (Rennie and Correia 1994) or I_K,L (Rüsch and Eatock 1996). In our type I hair cells, I_K,L usually begins activating 5-15 mV more negative than our standard holding potential, V_H = 67 mV. Such type I hair cells show large instantaneous currents on being stepped from V_H (Fig. 2, A and D). The instantaneous current, which we refer to as I₆₇, is deactivated when the cell is hyperpolarized beyond 80 mV. In some cases, almost all of I_K,L is activated at V_H so the response to depolarizing steps is dominated by the instantaneous current (Fig. 2, B and E). More typically, depolarizing steps evoke both the instantaneous component and a slow, sigmoidally activating component (Fig. 2, A and D). In type I hair cells from other organs, outwardly rectifying currents besides I_K,L may contribute to the slow, sigmoidally activating currents (Masetto et al. 2000; Rennie and Correia 1994; Rüsch and Eatock 1996; Rüsch et al. 1998). In the turtle, however, most conductance-voltage curves from type I cells would appear to be dominated by a single, I_K,L current. Given this interpretation, we attribute the instantaneous current to I_K,L channels active at V_H and the sigmoidal component to additional I_K,L channels becoming active with depolarizations above V_H.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Voltage-clamp records (top) and current-voltage (I-V) curves (bottom) for 3 type I hair cells having IK,L currents. A and D: the current is only partly activated at the holding potential, VH = 67 mV. As a result, there is an instantaneous current at the start of all voltage steps. Depolarizing steps result in a slow, sigmoidal activation of additional current, whereas hyperpolarizing steps deactivate the instantaneous current. Both activation and deactivation can be seen in the I-V curves as departures of the late current from the early current. B and E: most of IK,L is activated at VH. A large instantaneous current is seen. Hyperpolarizing steps deactivate the current; depolarization steps result in only a slight activation. The late I-V curve shows a large departure from the early curve only for hyperpolarizing steps. C and F: the current is activated at voltages more depolarized than VH. The instantaneous current is small. Slow, sigmoidal activation is seen during depolarizing steps. There is no deactivation for hyperpolarizing steps nor is there any evidence of inwardly rectifying currents. In all panels, voltage steps are 200-ms duration from a holding potential, VH = 67 mV, to voltages in 10-mV steps from 137 to 7 mV. In Figs. 4-7, currents were measured 0.5 or 1 ms (early) and 195-200 ms (late) after the start of the step; voltages are corrected for series resistance. The horizontal and vertical lines for each I-V curve indicate zero current and the resting potential, respectively.

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View this table: [in this window] [in a new window]   Table 1. Correlation between hair-cell morphology and the presence of an instantaneous (I67) conductance at 67 mV

View larger version (26K): [in this window] [in a new window]   Fig. 3. Voltage-clamp records (top) and current-voltage (I-V) curves (bottom) for 3 type II hair cells harvested from the peripheral zone near the planum (A and A1), near the torus (B and B1), or from the central zone (C and C1). All 3 cells have outwardly rectifying currents that activate between 57 and 47 mV. The 2 cells from the peripheral zone have fast activation kinetics, while the central cell has slow activation kinetics. Inactivation in most conspicuous in the planum cell, while inwardly rectifying currents are largest in the torus cell.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Scatterplots relating the half-activation times of outwardly rectifying currents with the magnitude of the associated conductance (A) and the amount of inactivation (B). Half-activation times and inactivation index = (1-Iend/Ipeak) were measured during 200-ms, 30-mV depolarizing steps from a holding potential, VH = 67 mV. Ipeak was the maximal current during the step and Iend was the average current during the last 5 ms of the step. Conductance was measured from the instantaneous current obtained at the end of a step to 52 mV after the voltage was held at 7 mV for 200 ms; residual capacitative current, as estimated from extrapolation of tail current, was subtracted. There is a trend for currents with slower activation kinetics to be larger and to show less inactivation. Data from type I and II hair cells from different regions of the neuroepithelium (see key) are consistent with these trends. Data in each panel were fit to power laws, y = axb, where x is half-activation time in milliseconds. In A, y is conductance in nS, a = 2.20 ± 0.47, and b = 0.97 ± 0.08; in B, y is inactivation index, a = 1.20 ± 0.26 and b = 1.26 ± 0.08.

ACTIVATION RANGE. Outwardly rectifying currents from type II hair cells usually require depolarizations to between 50 and 60 mV to begin activating (Fig. 3). In contrast, most type I hair cells are already partly activated at our holding potential (V_H = 67 mV) or just above it (Figs. 2 and 7F).

ACTIVATION KINETICS. We measured half-activation times during 200-ms depolarizations to 37 mV. For type I hair cells, half activation typically took 20-100 ms (Figs. 2 and 4A; Table 2).

CONDUCTANCE MAGNITUDES. These were estimated from the instantaneous currents when cells were stepped to 52 mV after being depolarized to 7 mV for 200 ms. Outward currents evoked by this depolarization are relatively large in type I hair cells (Fig. 2). The corresponding conductances are typically 100-300 nS and are similar in type I cells with or without I₆₇ (Fig. 4A, Table 2). Currents and the associated conductances are smaller in type II hair cells from the planum (15-25 nS) and even smaller in type II hair cells from the torus (5-15 nS).

INACTIVATION. This was measured as the proportional decline from the peak current to the current evoked at the end of a 200-ms depolarization to 37 mV. Inactivation was correlated with t_1/2. Declines were negligible in type I hair cells and in slow type II central cells but were present in many fast type II cells from both parts of the PZ and from the CZ. The inactivation index seldom exceeded 0.5, suggesting that even cells showing inactivation had a noninactivating current as well.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Inactivation of outwardly rectifying currents in 2 peripheral-zone type II hair cells with fast activation kinetics, one near the planum (A and A1) and the other near the torus (B and B1). A and B: cells were stepped from VH = 67 to a variable conditioning voltage for 200 ms and then to a test voltage of 3 mV for 200 ms. Conditioning voltage ranges from 127 to 27 mV as indicated by numbers on both conditioning and test responses. Fast inactivation is seen in A but not in B as a decline in the peak current as the conditioning step is made progressively more positive. A1 and B1: the amplitude of the transient and sustained components of the test response are plotted as a function of the preceding conditioning voltage. The sustained current was measured as the average current during the last 5 ms of the test pulse to 3 mV. For the transient component, the sustained current was subtracted from the peak current during the test pulse. There is a large transient component indicative of fast inactivation in A1 but not B1. Despite the difference in fast inactivation for the 2 cells, both show a substantial change in the sustained response as a function of the voltage of the immediately preceding conditioning pulse.

INWARDLY RECTIFYING CURRENTS. Most (>90%) type II cells had inward currents that activated negative to 87 mV. In hair cells from the frog sacculus (Holt and Eatock 1995), two inward currents (I_K1 and I_h) were distinguished by their activation kinetics, ion selectivity, and sensitivity to divalent cations. In the present study, we relied on activation kinetics to recognize the two currents. As was the case in other hair cells (Eatock et al. 1998; Holt and Eatock 1995; Weng and Correia 1999), I_K1 has rapid monoexponential kinetics and is fully activated by 10-25 ms (Fig. 6A). At very large negative voltages near 137 mV, I_K1 declines with time, suggestive of a multi-ion block (Hille 1992). Many cells had a mixture of I_K1 and I_h; this resulted in a rapid activation followed by a slow sigmoidal activation of inward current (Fig. 6B). Table 3 summarizes the incidence of the two currents in type II hair cells. Cells with a mixture of I_K1 and I_h and those only having I_K1 each occurred in slightly less than half the cases. Only a few cells had I_h but not I_K1.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Inward currents in type II hair cells. A and B: voltage-clamp records from 2 peripheral-zone type II cells. Vertical lines indicate times chosen to separate inward currents in response to a 107-mV step into IK1 and Ih components. The cell in A, located near the torus, has a single (IK1) inward component, which is completely activated by 25 ms. For the cell in B, located near the planum, there are 2 inward components, a fast-activating IK1 and a slow-activating Ih. In A and probably in B, the IK1 current declines with time during the step to 137 mV.

Biophysical properties of IK,L

ACTIVATION KINETICS. To study the activation kinetics of I_K,L, voltage was first stepped for 200 ms from V_H to 127 mV to deactivate the current. This was followed by a 500-ms step to potentials ranging from 97 to 7 mV in 5-mV steps. In the example of Fig. 7, A and B, activation is first seen at 77 mV. The increase in outward current has a sigmoidal time course which can be fit by the difference of two exponentials (Fig. 7B)

(1)

an equation consistent with a kinetic scheme C₂  C₁  O involving two closed (C₂ and C₁) and one open (O) state. No attempt was made to fit the small decline in current seen at potentials larger than 30 mV. Fits are good for potentials between 77 and 47 mV; small discrepancies at more positive potentials may reflect the increasing voltage errors arising from uncompensated series resistance. The slow time constant, ₁, declines 20-fold with voltage, from 300 ms at 72 mV to 15 ms at 22 mV (Fig. 7C, - ). When plotted logarithmically, there is a slightly larger proportional decrease in the fast time constant, ₂ (Fig. 7C,  - ). Similar trends are seen in time constants averaged for 10 hair cells (Fig. 7D).

View larger version (28K): [in this window] [in a new window]   Fig. 7. Activation of IK,L current in type I hair cells. A: voltage-clamp records for a hair cell. IK,L is deactivated by a 200-ms step to 127 mV from a holding potential of 67 mV. The current is then activated by a 500-ms step to voltages between 97 and 17 mV. This is followed by a step to 52 mV to measure tail currents. B: activation is first seen at 77 mV and becomes faster with larger depolarizations. Kinetics well fit by text Eq. 1 () until 37 mV, where there are small discrepancies. C: time constants, obtained from the nonlinear fits of text Eq. 1 to the points in B, are plotted as a function of voltage. Both time constants decline as voltage increases. D: mean values of the time constants are plotted as a function of voltage for 10 type I cells; bars indicate SE. E: tail currents extrapolated to the beginning of the step to 52 mV and an empirically determined reversal potential of 84.3 mV were used to estimate conductances at the end of the preceding step for the cell illustrated in A-C. Curve is fit by a Boltzmann equation (Eq. 3). No attempt was made to fit the decline in conductance for depolarizations beyond 34 mV. F: normalized conductances are shown for 35 type I hair cells, including 28 cells (· · ·) with and 7 cells () without an instantaneous (I-67) current. Thicker dashed curve (- - -) is the activation curve of a central type II cell that had a typical activation curve. Five of the 7 type I cells without I-67 have activation curves that fall between the curves for type I cells with I67 and the type II cell.

ACTIVATION RANGE. Conductances at the end of the 500-ms voltage steps were derived from extrapolated tail currents (I_TAIL) obtained at 52 mV (V_TAIL; Fig. 7E). The formula

(2)

was used. The leak current (I_LEAK = 0.31 nA) was obtained by taking the average tail current between 87 and 97 mV, and the reversal potential (V_K = 84.3) was estimated as described in REVERSAL POTENTIAL AND ION SELECTIVITY. g_K,L began increasing near 80 mV, reached a maximum of 119 nS at 37 mV, and then declined by 9.3% over the next 14 mV. The decline seen with large depolarizations may reflect an inactivation of g_K,L or the extracellular accumulation of K⁺ ions during large, prolonged outward currents (Rennie and Correia 2000). Such an accumulation would decrease the driving force (V_TAIL  V_K), which would explain why there is a larger decline in the tail currents than in the currents during the preceding voltage step. Data points between 97 and 34 mV were fit by a Boltzmann equation

(3)

with g_MAX = 118.0 ± 2.0 nS, V_1/2 = 65.9 ± 0.3 mV, and V_S = 5.7 ± 0.3 mV.

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DEACTIVATION OF I_K,L. To study the kinetics of I_K,L at hyperpolarized potentials, we used a deactivation protocol (Fig. 8A). The cell was first stepped to 57 mV to increase activation of I_K,L and then to potentials ranging from 67 to 127 mV to deactivate the current. Deactivation becomes faster with increasing hyperpolarization. As a result, the traces for more negative voltages cross those for less negative voltages (Fig. 8, A and B). Deactivation cannot be fit with a single exponential. Rather, a sum of two exponentials

(4)

is needed (Fig. 8B). Although this might suggest the presence of two distinct currents, it is also to be expected from a three-state C₁  C₂  O model provided that the hyperpolarizing step does not result in a complete steady-state deactivation. Figure 8C plots the fast and slow time constants as functions of membrane potential. Included are the results of an activation analysis as well as the deactivation analysis. Over the voltage range from 127 to 87 mV, _SLOW increases from 20 to nearly 200 ms, while _FAST only increases from 7 to 11 ms. Over the same range, the relative magnitude of the fast component declines so that the ratio of I_FAST to I_SLOW in Eq. 4 falls from 1.1 at 127 mV to 0.2 at 87 mV.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Deactivation of IK,L in type I hair cells. A: in this cell, the voltage was stepped from VH = 67 to 57 mV for 200 ms to activate additional IK,L. This was followed by a hyperpolarizing step to various voltages, ranging from 137 to 67 mV in 10-mV steps. B: responses to hyperpolarizing steps in A are fit by a double exponential (). Also shown is the slow component of the double-exponential fit (- - -). Curves are displaced so that they each reach 0 for long times. C: time constants from the double-exponential fit are plotted vs. voltage. Also included are the corresponding time constants obtained from an activation analysis similar to that shown in Fig. 7. D: points are mean deactivation and activation time constants for a sample of 8 cells; bars are SE.

REVERSAL POTENTIAL AND ION SELECTIVITY. The reversal potential (V_REV) for I_K,L was determined as the intersection between two instantaneous I-V curves, one with the current partially activated and the other with it deactivated. A similar method has been used to characterize other currents (Adams et al. 1982). V_REV for I_K,L was obtained in four type I cells in our standard (4 mM K⁺) bath and when the cells were washed in local streams containing 4 and 12 mM [K⁺] solutions. Mean values were 83.3 ± 3.7 (SE) mV (bath), 86.9 ± 3.6 mV (4-mM wash), and 58.1 ± 0.9 mV (12-mM wash). Results are consistent with I_K,L being K⁺ selective. V_REV in the 4-mM wash was close to the calculated K⁺ equilibrium potential, V_K = 89.2 mV. Raising external [K⁺] to 12 mM shifted V_REV by 28.8 ± 3.1 mV, near the value of 27.9 mV calculated from the Nernst equation for K⁺. There was a negative shift of 3.6 ± 1.4 mV in the 4-mM wash. This could be explained if, in the absence of the local stream, there is an accumulation of K⁺ around type I cells, increasing [K⁺] concentration from 4 to 4.6 mM.

67

Pharmacology of I_K,L

Previous studies had characterized the effects of several external blockers on the I_K,L current in type I hair cells harvested from the gerbil and pigeon cristae (Rennie and Correia 1994) or recorded from explants of the neonatal mouse utricle (Rüsch and Eatock 1996). We wished to determine if I_K,L had a similar pharmacology in type I hair cells from the turtle posterior crista. Although our results are consistent with those previously reported, some of our interpretations are different. In particular, we found that the actions of some external blockers can only be appreciated by a complete activation analysis.

4-AP. I_K,L was blocked by low concentrations of 4-AP in a state-dependent manner. This is illustrated by a type I hair cell studied with our usual activation protocol (Fig. 9, A and B). A 30-µM dose almost completely blocked the current at 67 mV but had proportionately smaller effects as depolarization was increased to 7 mV. The result is a 10- to 15-mV shift in the activation curve (Fig. 9C) with only a small (25-30%) reduction in g_MAX. Other effects include an approximately twofold slowing of activation kinetics (Fig. 9D) and a reduction in the inactivation produced by large depolarizations (Fig. 9B). With the exception of the reduction in g_MAX, effects are consistent with a so-called reverse-use dependent block in which the blocker only attaches to the channel in the closed state and prevents transitions to the open and inactivated states (Remillard and Leblanc 1996).

View larger version (21K): [in this window] [in a new window]   Fig. 9. IK,L is blocked in a reverse-use dependent manner by low concentrations of 4-aminopyridine (4-AP). Data are from a type I hair cell. Protocol as in Fig. 7. A and B: voltage-clamp records before (A) and during (B) application of 30 µM 4-AP, showing that 4-AP completely blocks activation of IK,L at 67 mV but has progressively less effect for depolarizing steps between 47 and 7 mV. C: the voltage-dependent effects of 4-AP application result in a 13-mV shift in the activation curve. There is a nearly complete return to control conditions on drug removal. Plotted is the normalized conductance obtained by measuring tail currents (for details, see legend to Fig. 7E). Conductances were normalized by subtracting a leak current from the tail current and dividing the result by gMAX, the maximal conductance obtained by a fit of text Eq. 3. The key also pertains to D. D: in addition to the shift in the activation curve, 4-AP results in a slowing of activation kinetics at a given voltage. Fast (top) and slow time constants (bottom; see text Eq. 1) are plotted vs. voltage. E: dose-response curves based on data from 9 type I and 12 type II cells. Four type I cells and 1 type II cell were studied at multiple concentrations. Curves are Michaelis-Menton fits.