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Departments of
¹ Ophthalmology
² Physiology, Asahikawa Medical College, Asahikawa 078-8510, Hokkaido, Japan

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
In the ciliary muscle, the tonic contraction requires a sustained influx of Ca²⁺ through the cell membrane. However, little has hitherto been known about the route(s) of Ca²⁺ influx in this tissue that lacks voltage-gated Ca²⁺ channels. To identify ion channels as the Ca²⁺ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. Experiments were carried out using pipettes filled with K⁺-free solution containing 100 mM caesium aspartate, 5 mM BAPTA and 180 µM GTP (pH 7.0; the intracellular free Ca²⁺ concentration, [Ca²⁺]_i= 70 nM). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li⁺, Na⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺, estimated by cation replacement procedures, were 0.9: 1.0: 1.5: 0.2: 0.3: 0.4: 0.5 for NSCCL, and 1.0: 1.0: 1.8: 2.5: 2.6: 3.2: 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca²⁺]_i. Both NSCCL and NSCCS were dose-dependently inhibited by 1–100 µM SKF96365 La³⁺ and Gd³⁺, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca²⁺ entry pathway required for sustained contraction of the bovine ciliary muscle. RT-PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNAs of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated cation channels.

(Received 1 April 2004; accepted after revision 15 July 2004; first published online 22 July 2004)
Corresponding author A. Takai: Department of Physiology, Asahikawa Medical College, Asahikawa 078-8510, Hokkaido, Japan. Email: takai{at}asahikawa-med.ac.jp

	Introduction

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The ciliary muscle, an intraocular muscle responsible for visual accommodation and regulation of aqueous humour outflow, is densely innervated by cholinergic nerve fibres, and its contraction is initiated and sustained by stimulation of muscarinic receptors on the surface of the muscle cell membrane by the transmitter acetylcholine (Glasser & Kaufman, 2003). In many other mammalian smooth muscles, contraction induced by muscarinic stimulation has long been known to be accompanied by a depolarization concomitant with an increase in the conductance of the cell membrane, and this is usually attributed to the opening of cation channels with low ion selectivity, termed ‘receptor-operated’ non-selective cation channels (NSCCs) (Bolton, 1979; McFadzean & Gibson, 2002). Depolarization in response to muscarinic stimulation has also been demonstrated by the intracellular microelectrode method in dog ciliary muscle (Ito & Yoshitomi, 1986) and in a human ciliary muscle cell line (Korbmacher et al. 1990). In previous experiments we have examined the effects of a cholinergic agonist, carbachol (CCh), on the membrane potential and current in smooth muscle cells freshly isolated from the bovine ciliary body, using the whole-cell clamp method (Takai et al. 1997). We have confirmed thereby that, under current clamp at 0 pA, CCh causes an atropine-sensitive depolarizing response which is concurrent with an increase in the membrane conductance. We have also shown that, under voltage clamp, CCh evokes a current which is resistant to organic Ca²⁺ channel antagonists and reverses the polarity at a holding potential near 0 mV (Takai et al. 1997). These previous observations strongly suggest that muscarinic stimulants activate some type(s) of NSCC to produce the electrical phenomena in the ciliary muscle.

However, our knowledge about the channels in the ciliary muscle is still very limited. For example, no experimental evidence has hitherto been available to determine whether muscarinic stimulation activates a single species of NSCC or more than one type of NSCC. Although the polarity reversal at a potential near 0 mV is indicative of a low ion selectivity, quantitative comparison of the relative permeabilities of the channels to cations has not been performed. Also, very little is known about the functional roles for the channels. Even if the opening of the channels causes a depolarization of the muscle cell membrane, it has been shown that depolarization by itself cannot initiate or maintain the contraction of the ciliary muscle (Suzuki, 1983).

In the present experiments, as a continuation of our previous study on the bovine ciliary muscle, we have further examined the properties of the currents evoked by superfusion of CCh under whole-cell voltage clamp. Since transient receptor potential (TRP) channel homologues are now considered as possible molecular candidates for receptor-operated NSCCs (see Clapham et al. 2001; Minke & Cook, 2002; Inoue et al. 2003), we have also examined the existence of their mRNAs in the ciliary muscle by RT-PCR. By analysing the noise of the currents evoked by CCh we have obtained results that indicate that, in most ciliary muscle cells, muscarinic stimulation activates two types of NSCC (NSCCL and NSCCS) with widely different single-channel conductances (35 pS and 100 fS). These channels differ from each other with regard to the activation kinetics as well as in the relative permeabilities to alkali and alkaline earth metal ions, and are likely to be linked to muscarinic receptors by different signalling pathways. We also show that several substances that inhibit the CCh-evoked NSCC currents also inhibit the tonic component of the contraction of ciliary muscle bundles induced by CCh without strongly affecting the initial phasic component. NSCCL and/or NSCCS, either of which admits Ca²⁺, may act as a pathway for Ca²⁺ entry required for sustained contraction of the ciliary smooth muscle. In the RT-PCR experiments we have detected at least four types (TRPC1, TRPC3, TRPC4 and TRPC6) of TRP mRNA in the bovine ciliary muscle.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Solutions and chemicals

The normal physiological saline solution (PSS) used was of the following composition (mM): NaCl, 127; KCl, 5.9; CaCl₂, 2.4; MgCl₂, 1.2; glucose, 11.8; Hepes, 10. The pH was adjusted to 7.4 (at 30°C) by titration with NaOH. In the cation substitution experiments, NaCl, KCl, CaCl₂ and MgCl₂ in PSS were totally replaced with an isosmotic concentration of an alkaline metal salt (LiCl, NaCl or CsCl; 138 mM) or an alkaline earth metal salt (MgCl₂, CaCl₂, SrCl₂ or BaCl₂; 92 mM). High K⁺ (100 mM) PSS was prepared by replacing NaCl with equimolar KCl.

Unless otherwise mentioned, pipettes for whole-cell clamp experiments were filled with a solution containing (mM): NaCl, 5; caesium aspartate, 100; MgCl₂ 5; ATP (disodium salt), 5; Hepes, 20; 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 5; and GTP, 0.18. The pH was adjusted to 7.0 with NaOH. The free Ca²⁺ concentration was adjusted by changing the Ca/BAPTA ratio. The apparent dissociation constant of BAPTA for Ca²⁺ was assumed to be 0.35 µM (Takai et al. 1997). Pipettes used for on-cell patch clamp experiments were filled with normal PSS.

Carbamylcholine chloride (carbachol; CCh), atropine sulphate, GTP, guanosine 5'-O-(3-thiotriphosphate) (GTPS), pertussis toxin and cholera toxin were purchased from Sigma Chemical Co. (St Louis, MO, USA). BAPTA was obtained from Dojindo Laboratories (Tabaru, Kumamoto, Japan). Iberiotoxin (synthetic) was a product of Peptide Institute, Co. (Osaka, Japan). All other chemicals used were of analytical grade.

Experiments were carried out at 30°C, unless otherwise mentioned.

Preparation of ciliary muscle cells

Bovine eyes were enucleated soon after the animals were killed in a local slaughterhouse and transported to our laboratory in ice-cooled PSS. The eyes were incised circumferentially about 5 mm posterior to the limbus, and after the vitreous humour and lens were removed, the ciliary muscle was carefully dissected out from the scleral spur. Single muscle cells were obtained by the method previously described (Takai et al. 1997). Briefly, the excised muscle was equilibrated in nominally Ca²⁺-free PSS containing 100 mM K⁺ at 35°C for 30 min and transferred to a solution of the same composition supplemented with 0.4% (w/v) collagenase (Wako Junyaku Co.) and 0.02% (w/v) papain (Sigma). After incubation for 20–25 min, the cell suspension was filtered with a fine nylon mesh. The dispersed cells were collected by centrifuging at 200 g for 2 min and then resuspended in nominally Ca²⁺-free, high K⁺ (100 mM) PSS.

Electrical recordings

An Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) was used for voltage clamp experiments. The borosilicate glass pipettes used had a resistance of 13.5 ± 0.3 M (n= 49) when filled with the pipette solution for whole-cell voltage clamp. For command pulse generation and digital data acquisition we used a Digidata 1200B interface controlled by the pCLAMP software (version 9; Axon Instruments) running on a Windows-based computer. Relatively short sections of data were recorded on a magnetoptic disk after on-line digitization. Long sections of data were recorded on a video cassette using an RP-880 PCM converting system (NF Electronic Instruments, Tokyo, Japan). Mathematical processing and analysis of data were carried out using the ClampFit software (version 9; Axon Instruments, Foster City, CA, USA) and the Origin software (version 7.5; OriginLab, Northampton, MA, USA).

Noise analyses

Non-stationary variance analysis.  The current signal was filtered by an analog 4-pole Bessel filter at 1 kHz (–3 dB) and digitized at 2 kHz, before the mean µ and the variance ² around the mean were calculated for every 500 sample points.

The unitary current amplitudes were estimated from the slopes of the best-fit lines obtained by linear regression of (µ, ²) data, assuming the following relationship (see, e.g. Sigworth, 1980)

(1)

where denotes the unitary current amplitude and ₀² represents the contributions from undesired fluctuations such as thermal noise in the voltage clamp. For this purpose we selected CCh-evoked responses such that their µ–² relationships could be fitted by single straight lines (see Fig. 2). The unitary chord conductances were then calculated, using the value of estimated as the slope of the regression line, as

(2)

where is the unitary conductance, E_h is the holding potential and E_r is the zero-current potential (i.e. the potential of polarity reversal).

View larger version (29K): [in this window] [in a new window]   Figure 2.  Inward currents evoked by CCh Whole-cell voltage clamp recordings similar to those shown in Fig. 1. At a holding potential (Eh) of –50 mV, 2 µM CCh was applied to the bath for 30 s. A, whole-cell currents evoked by CCh. Four typical responses (a–d), recorded from four different cells, are shown. Note the marked difference in the noise level and mean amplitude of the currents. B, non-stationary variance analysis. The four records shown in A were low-pass filtered at 1 kHz (–3 dB, 4-pole Bessel), digitized at the sampling rate of 2 kHz, and then the mean current (µ, in pA) and variance (2, in pA2) were calculated for every 500 data points. The linear appearance of the µ–2 relationships in Ba and Bd indicates that the CCh-evoked currents were produced by the opening of either NSCCL (a) or NSCCS (d) alone. The slope of the regression line was –1.7 pA in Ba (dot–dash line) and –4.8 fA in Bd (dashed line), from which the unitary conductance was estimated, assuming the reversal potential, Er= 0 mV, to be 35 pS and 96 fS, respectively. In Bb and Bc, lines of the same slopes (1.7 pA and 4.8 fA) are fitted by eye with the two apparently linear components, which are probably produced by the simultaneous opening of NSCCL and NSCCS. See text for further details.

(3)

where N is the number of the channels and p is the non-stationary open-state probability (Sigworth, 1980). Equation (1) is a limiting case of eqn (3) where p 0.

Spectral analysis.  The signals from the cassette tape were digitized at 5 kHz and digitally high-pass filtered at 0.2 Hz and low-pass filtered at 2 kHz (–3 dB, 8-pole Butterworth). These band-passed noise data were divided into segments each containing 4096 or 8192 data points, for which two-sided power spectral densities were calculated by fast Fourier transform and then averaged to obtain a mean noise power spectrum. The net two-sided spectral density for agonist-induced currents was obtained by subtracting the mean spectrum of noise in the absence of agonist from that in its presence.

The positive-frequency halves of the symmetrical two-sided net spectra were fitted by non-linear least-squares method (Levenberg-Marquardt algorithm) with the model function defined as the sum of two Lorentzian components:

(4)

where S(f) is the two-sided power density at the frequency f, S₁(0) (> 0) and S₂(0) ( 0) are the zero-frequency asymptotes, and f₁ and f₂ (0 < f₁ < f₂) are the corner frequencies. If all noise components of an agonist-induced current are detected in the frequency domain as the sum of two Lorentzians, then we will have

(5)

where _c² denotes the variance of the agonist-induced current in the time domain (Cull-Candy et al. 1988; Howe et al. 1988).

Some (but not all) power spectra with two Lorentzian components can be simulated using a simple three-state reaction model (Colquhoun & Hawkes, 1977):

where A is the agonist, and R and R* denote the closed channel and the open channel, respectively, which are complexed with the receptor. Now, assign the molar concentration of the agonist to c and write the spectral function in the form of eqn (4); then, for this particular model, f₁, f₂, S₁(0) and S₂(0) are given as explicit functions of the rate constants, k₁, k₂, and ß, as follows (see Colquhoun & Hawkes, 1977):

(6)

(7)

(8)

and

(9)

where

and

The equilibrium probabilities, p_AR*, p_AR and p_A+R, that the channel occupies each of the three states, AR*, AR and A + R, are also explicitly given, as functions of c, by the following set of equations:

(10)

Note that p_AR*+p_AR+p_A+R= 1.

The dose–activation relationship for the agonist is related to p_AR* (=p_AR*(c)) by

(11)

where (c) is the fractional intensity of the response at c, and K is the apparent dissociation constant defined by

(12)

Obviously, the use of the reaction model (Scheme 1) is justified only when there exists a set of physico-chemically realistic values of k₁, k₂, and ß which gives a reasonable fit of the spectral function to data. In the present experiments we tried to estimate such values by numerically solving the eqns (6)–(9) simultaneously in terms of k₁, k₂, and ß with the use of the values of f₁, f₂, S₁(0) and S₂(0) determined by directly fitting the model function eqn (4) to the spectral data. For this purpose, we used the Mathematica software (version 5.0; Wolfram Research, Champaign, IL, USA). We also tried to fit the spectral data by the non-linear least-squares method with the model function eqn (4) after making the substitutions defined by eqns (6)–(9), and thereby estimate the values of k₁, k₂, and ß.

Permeability ratios

The ratio of the permeability of a monovalent or bivalent cation to that of Na⁺ was estimated from the shift of reversal potential caused by total substitution of Na⁺, K⁺, Ca²⁺ and Mg²⁺ in the bath solution with the cation, using the following equation (Lewis, 1979):

(13)

where X represents the substitute ion (which may be Na⁺ itself), z (= 1 or 2) is the valency of X, [X]_o is the extracellular concentration of X, P_X is the permeability coefficient for X, E_X is the reversal potential measured using X as the substitute, and F, R and T have their usual thermodynamic meanings.

Mechanical recordings

The methods of tension recording and of superfusion were essentially the same as previously described (Ashoori et al. 1985). Briefly, a small piece (about 1 mm wide and 5 mm long) of smooth muscle was carefully dissected from an excised ciliary muscle bundle. The preparation was vertically mounted in a small organ bath (0.5 ml in volume) through which solution flowed at a constant rate of 2 ml ml^–1. The tension was isometrically recorded using a U-gauge transducer (type UL-2GR; Minebea Co., Tokyo, Japan) and a potentiometric pen-recorder.

RT-PCR experiments

A QuickPrep mRNA purification kit (Amersham Biosciences, Little Chalfont, UK) was used to isolate polyadenylated mRNA from the bovine ciliary muscle (whole tissue; 10–50 mg). The polyadenylated mRNA obtained was transcribed to first-strand cDNA using a PowerScript reverse transcriptase (BD Biosciences, San Jose, CA, USA) and a 5'-d(T)₂₅N_–1N-3' primer (where N = A, C, G or T; and N_–1= A, G or C). GeneAmp PCR System 9700 thermal cyclers (Applied Biosystems, Foster City, CA, USA) were used to amplify transient receptor potential cannonical (TRPC) sequences with Advantage 2 polymerase (BD Biosciences) and the gene-specific primer pairs (see Table 2) which were designed, based on the reference sequences (see Table 2) obtained from the GenBank database. Either a two-step or a three-step PCR protocol (with 30–45 thermal cycles) was chosen, depending on the theoretical melting temperature for each primer pair. The products of the PCR reactions were immediately subjected to electrophoresis in agarose gel (0.9–1.2%). The gels were stained with SYBR Gold fluorescent dye (Molecular Probes, Eugene, OR, USA) after electrophoresis and visualized with a DarkReader visible light transilluminator (Clare Chemical Research, Dolores, CO, USA). Amplified DNA fragments of expected sizes were routinely extracted from the gel and subcloned into a pCR4-TOPO sequencing vector (Invitrogen). The vector was transformed into chemically competent DH5E. coli cells which were then cultured after colony selection using ampicillin as the marker. The nucleotide sequence of the insert in the plasmid isolated from the culture was analysed by a Long-Read Tower sequencer (Amersham Biosciences) after thermal cycling reaction with ThermoSequenase DNA polymerase and CyDye terminators (Amersham Biosciences). For primer pairs which gave no clear specific amplification in initial trials, we examined the effects of changing the annealing temperature in a ± 5°C range around the value suggested by the Oligo software (Molecular Biology Insights, Cascade, CO, USA) using a gradient thermal cycler (MasterCycler Gradient, Eppendorf, Hannover, Germany), and thereby tried to find appropriate amplification conditions.

View larger version (82K): [in this window] [in a new window]   Figure 13.  Detection of TRPC transcripts in bovine ciliary muscle by RT-PCR The existence of TRPC mRNAs in the bovine ciliary muscle (whole tissue) was examined by RT-PCR. A typical agarose-gel image taken after electrophoresis of the PCR products is shown (brightness inverted). Each PCR reaction mixture contained 10–18M cDNA as the template. The PCR reactions with the five positive-control primer pairs and four bovine TRPC-specific primer pairs yielded DNA fragments of the expected sizes (in parentheses; unit, bp): GAPDH (540), 5'actin (1000), 3'actin (720), 6 kb clathrin (570), 2 kb clathrin (550), TRPC1 (259), TRPC3 (323), TRPC4 (382) and TRPC6 (321). The bands of the amplified TRPC DNA fragments are indicated by arrowheads. The results of the sequence analysis using the DNA fragments extracted from the bands have been deposited in the GenBank under the accession numbers which are given in Table 2 together with the sequences of the primer pairs used. The TRPC3-specific primers (not listed in Table 2) used in this PCR experiment were 5'-CCAGCAGCAGCTCTTGAC-3' (forward) and 5'-ACTGTGTGGTTTTCACCCTG-3' (reverse), which amplify an initial 323 bp segment of the 1071 bp fragment (AB179743) listed in Table 2. The primers for TRPC2 (expected size in bp, 206), TRPC5 (275) and TRPC7 (830) gave no specific amplifications.

To describe dose–response relationships, we generally used the Hill function

(14)

where (x) is the intensity of the response in the presence of an activator or inhibitor whose concentration is x, _max is the maximal or control response, K denotes the apparent dissociation constant and h stands for the Hill coefficient (h > 0 for dose–activation relationships and h < 0 for dose–inhibition relationships). Note that eqn (11) is a special form of eqn (14). The values of K and h were estimated with standard errors by fitting this model function to the data by non-linear least squares regression, using as weight the reciprocal of the square of the standard error at each value of x.

Statistics

Significance tests for inference about the slope and the intercept of linear regression lines were carried out using the standard method of analysis of variance (Snedecor & Cochran, 1980). Comparison of two experimentally obtained values was made by a modified Student's t test (Snedecor & Cochran, 1980). Differences were taken as statistically significant when two-tailed probabilities less than 0.05 were obtained.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Two types of non-selective cation channels activated by CCh

Figure 1 shows typical responses to superfusion of 2 µM CCh recorded in bovine ciliary myocytes by whole-cell voltage clamp at 30°C and at holding potentials (E_h) from –50 to +40 mV. CCh evoked a clear inward current at E_h=–50 mV. The polarity of the current was inverted at voltages higher than 0 mV (Fig. 1A). This response to CCh was completely abolished by 0.1 µM atropine, whereas it was not affected by verapamil or by tetrodotoxin, confirming our previous observations (Takai et al. 1997). In 91% of the 2678 cells examined using pipettes filled with a solution containing 180 µM GTP, the response to CCh was accompanied by a marked increase in noise (see also Fig. 2Aa–c). In most of these cells, discrete current steps of a constant amplitude could be readily distinguished at the onset of CCh application or during its removal, where the number of channels being activated was small (Fig. 1B). The plot of the step size against E_h crossed the abscissa at a potential close to 0 mV (Fig. 1C), indicating that the current steps were produced by openings of a non-selective cation channel (NSCC). From the slope of the E_h–I relationship (Fig. 1C), the unitary conductance of the channel was estimated at 36.0 ± 0.3 pS (n= 58 cells). We refer to this type of NSCC with a relatively large unitary conductance as NSCCL.

View larger version (19K): [in this window] [in a new window]   Figure 1.  Whole-cell voltage–current relationship In a smooth muscle cell freshly isolated from the bovine ciliary body, whole-cell currents were recorded at 30°C by voltage clamp using a pipette filled with a solution containing 180 µM GTP. A, currents elicited by application of CCh (2 µM) were recorded at six different holding potentials (Eh). CCh was applied to the bath at 5 min intervals. Note the polarity reversal at about 0 mV. B, single-channel currents produced by superfusion of 2 µM CCh. These recordings were taken during wash-off of the CCh so that the number of channels being activated is small. C, the Eh–I relationship for the current shown in B. The unitary slope conductance is estimated at 36 pS. See text for further explanation.

In certain cell types it has been reported that chloride channels are activated by agonists of muscarinic cholinoceptors (Dickinson et al. 1992) or adrenoceptors (Amédée et al. 1990; Wang & Large, 1991). However, in our experiments, the contribution of chloride ions to the inward currents should be little, if any, because the inward current was clearly observed at membrane potentials around –50 mV, which is close to the equilibrium potential of chloride.

Figure 2 shows four typical patterns of the currents evoked by superfusion of 2 µM CCh under whole-cell voltage clamp at –50 mV. As noted above, the majority of the cells gave responses accompanied by a clear increase in noise (Fig. 2Aa–c). In these, the noise often started to appear before the change of mean current level became apparent (Fig. 2Ac; arrow). The intensity of noise, however, differed largely among the cells, and in many cells it did not appear to be directly correlated with the mean amplitude of the response. In the 241 other cells, CCh produced a relatively large inward current with no apparent increase in noise (Fig. 2Ad). In such cells, the current also changed polarity at a potential near 0 mV (see Fig. 7B). These observations strongly suggest that in most ciliary muscle cells CCh also opened a distinct species of NSCC with a unitary conductance much smaller than that of NSCCL, which we call NSCCS.

View larger version (9K): [in this window] [in a new window]   Figure 7.  Effects of substitution of the charge carrier on the NSCCS current A, whole-cell currents evoked by bath application of 2 µM CCh at a holding potential of –50 mV in a ciliary myocyte which showed a current purely attributable to NSCCS openings (cf. Fig. 2Ad) in response to the application of CCh (upper trace). The same concentration of CCh evoked a larger response accompanied by a slight but clear increase in noise (arrow) when Na+, K+, Ca2+ and Mg2+ were isosmotically replaced by 92 mM Ba2+ (lower trace; see Fig. 5 for a power spectrum for this type of response). Unitary current amplitudes of 4 fA and 16 fA are estimated from the linear µ–2 relationships (not shown) for the currents in PSS and Ba2+-substituted solution, respectively. To examine the voltage dependence of the current, a ramp voltage pulse falling from +120 to –80 mV at a constant rate of –1 V s–1 was applied before (p0) and during (p1) the applications of CCh. B, the voltage–current (Eh–I) relationships calculated as the difference between the responses to the ramp pulses (p1 minus p0). The current evoked in PSS showed inward-going rectification and changed the polarity at about –5 mV. The total cation substitution with Ba2+ caused a marked increase in the degree of rectification and a shift of the voltage of polarity reversal to about +25 mV.

µ–

µ–

In these cells the steady-state level of the whole-cell NSCCL currents at the holding potential of –50 mV was –8.6 ± 0.4 pA (n= 237 responses). Since the amplitude of the single-channel current has been estimated at 1.7 pA (see above), we calculate that only five channels were open on average at any one instant during the whole-cell responses. From the whole-cell capacitance (15.7 ± 0.3 pF; n= 187 cells), the mean surface area of the cells was estimated at 1.6 x 10³µm², assuming the specific membrane capacitance of 1.0 µF cm^–2. The density of NSCCL open during the response to CCh is therefore calculated to be as small as 0.003 µm^–2. This means that approximately one NSCCL is open per 300 µm² of surface membrane.

Figure 2Bd shows another example of a µ–² relationship obtained from a cell in which CCh produced an inward current with no clear increase in noise (see Fig. 2Ad). Linear least-squares regression fitted the data to a straight line having a slope of –4.8 ± 0.9 fA, which is very small in magnitude compared with the values obtained for the NSCCL currents (see above) but significantly different from zero (P < 0.001). This indicates that the CCh-evoked response attributable to the activation of NSCCS alone was accompanied by a slight but significant increase in whole-cell current fluctuations. Similar responses to CCh with smooth appearance were observed in 241 cells, and the µ–² relationships for these responses were fitted by single straight lines of the mean slope of –4.92 ± 0.04 fA (n= 331 responses), from which we estimated the value for NSCCS at 98 fS, assuming that E_r= 0 mV. At the holding potential of –50 mV, the mean whole-cell currents in these cells was –25 ± 4 pA (n= 331 responses), which was considerably larger in absolute value than the currents produced by NSCCL alone (see above). From these results the density of NSCCS open in the ciliary myocyte membrane during a response to CCh of smooth appearance is estimated at 3 µm^–2. This value is 1000 times the estimate given above for the density of NSCCL channels open during a response to the same concentration of CCh.

In more than 80% of the cells examined, the µ–² relationships of the CCh-evoked whole-cell currents could not be fitted with a single straight line. In most of these responses, however, discrete single-channel currents attributable to openings of NSCCL were clearly distinguished (see Fig. 1B), and the µ–² relationship could be fitted with a pair of straight lines having slopes of –1.7 pA and –4.9 fA (see Fig. 2Bb and c), which probably correspond to the contributions from NSCCL and NSCCS, respectively. Generally, if an agonist simultaneously activates two types of ion channel in a cell, the µ–² plot for the whole-cell current does not necessarily resolve into two components. But if NSCCL and NSCCS are coactivated by CCh, there are several factors that combine to make the contribution from each appear as separate components in the µ–² plane. (a) The time required for the response to stimulation by 2 µM CCh to reach a steady level was 5–10 s for NSCCS currents, whereas it was 10–20 s for NSCCL currents (compare the t–µ plots of Fig. 2Ba and d); thus, NSCCS currents tended to reach a steady level (let it be µ_s) faster than NSCCL currents. This would result in a predominant appearance of the NSCCS component in the µ range between 0 and µ_s in the µ–² plane. (b) NSCCS currents showed a steady level with very small fluctuations which lasted for 15–20 s during the stimulation by CCh for 30 s (see Fig. 2Bd). Openings of NSCCL during this steady period would give a clearly distinguishable component in the region where µµ_s(< 0). (c) The value of NSCCL (35–36 pS) was 360–380 times larger than that of NSCCS (96 fS) (see above). Reflecting this large difference in the unitary conductance levels, the contributions from NSCCL and NSCCS would appear in the µ–² plane as two linear components with largely different slopes. Thus it seems reasonable to interpret the two-component nature of the µ–² plots as a sign of the coexistence of NSCCL and NSCCS in most bovine ciliary myocytes. The absolute value of µ_s, which can be estimated from the abscissa of the intersecting point of the two linear components, was typically 60–80% of the maximal value of |µ| (|µ|_max; see Fig. 2Bb and c). From this percentage and the conductance ratio (360–380), the ratio of the numbers of NSCCS and NSCCL open during a typical response of the ciliary myocyte to CCh is roughly estimated at 500–2000.

Figure 3 shows the µ–² plot for a response evoked by 100 µM CCh in one of the relatively rare cells that gave a response, purely attributable to NSCCL openings alone, to a prior application of 2 µM CCh (see Fig. 2Aa and Ba). The large fluctuation of whole-cell currents due to the opening of NSCCL was also clearly observed at this agonist concentration which approaches the level required to produce the maximal response. Although the relationship between µ and ² now considerably deviated from linearity (Fig. 3), it could be fitted with eqn (3) under a constraint, = 1.75 pA, supporting the idea that was not largely changed from 35 pS by the elevation of the agonist concentration. From essentially similar fits of the curved µ–² relationships for the NSCCL currents evoked by 100 µM CCh in a total of six cells, a mean total channel number N of 83 ± 8 was obtained. Significant deviation from linearity was not detected for the µ–² plots of whole-cell NSCCS currents evoked by 100 µM CCh within the limits of the resolution of the present experiments (examined in 7 cells).

View larger version (18K): [in this window] [in a new window]   Figure 3.  Non-stationary variance analysis of NSCCL current evoked by high concentration of CCh In a cell in which 2 µM CCh invoked a response almost purely attributable to the opening of NSCCL as judged from the linear µ–2 relationship (not shown), NSCCL current was evoked by a higher concentration (100 µM) of CCh. At this agonist concentration, the relationship between µ and 2 considerably deviated from linearity. The continuous line is a best-fit of eqn (3) obtained by non-linear least-squares regression assuming a constant single-channel current amplitude (i.e. = 1.75 pA). From this fit, the total number of NSCCL present in the surface membrane of this cell, N, was estimated at 77. The line, 2=02+, which is tangent to the regression curve at µ= 0 pA, is also shown for comparison (dashed line). See the text for further details.

NSCCL.  Figure 4A shows examples of the noise spectra of the whole-cell currents purely attributable to NSCCL openings evoked in the same cell under voltage clamp at –50 mV in normal PSS by different concentrations of CCh (2, 0.5 and 100 µM; applied in this order at 5 min intervals).

View larger version (20K): [in this window] [in a new window]   Figure 4.  Power spectra of NSCCL currents A, power spectra of the noise of the whole-cell NSCCL currents evoked in the same cell at holding potential –50 mV by three different concentrations of CCh (2, 0.5 and 100 µM, applied in this order at 5 min intervals). The records were digitized at the sampling rate of 5 Hz and digitally high-pass filtered at 0.2 Hz and low-pass filtered at 2 kHz (–3 dB, 8-pole, Butterworth). The noise data were then divided into segments each containing 4096 samples, and the spectral densities were calculated for each segment by fast Fourier transform. Seventy-two control spectral densities and 36 spectral densities during the application of CCh were averaged. The net spectra are shown, obtained by subtracting the averaged spectral densities of control noise from that during superfusion of CCh. Only the positive frequency halves of the symmetrical two-sided spectra within the frequency range 1 Hz to 1 kHz are presented. The double Lorentzian function was fitted to the data in this range by non-linear least-squares regression (continuous line). The corner frequencies (f1 and f2) of the Lorentzian components (dashed lines) are indicated by arrows. Note the right–upward shift of the spectrum with increasing agonist concentration. Also note the disappearance of the second Lorentzian component in 100 µM CCh. The value of the corner frequency (75.8 Hz) of this spectrum obtained by regression to the single Lorentzian function (continuous line) was only slightly larger than the value of f1 (74.6 Hz) which was calculated together with that of f2 (196.2 Hz), assuming the three-state kinetics. Note the low level of the second Lorentzian component calculated on the same assumption (dash–dot line). The similarly calculated first Lorentzian component (not shown) was virtually identical to the regression curve. Ba, effects of changing the agonist concentration on the parameters of the power spectrum. The cells used were initially stimulated by 2 µM and then by one to three other concentrations of CCh for 30 s at 5 min intervals. The values of f1 (), f2 () and S1(0)/S2(0) (•) for the noise spectra of NSCCL currents evoked by various concentrations of CCh are plotted against the agonist concentration, c. Each symbol represents the mean of at least three values. Vertical bars indicating the standard errors of the means are omitted if within symbols. The graphs of f1, f2 (continuous lines) and S1(0)/S2(0) (dashed line) calculated based on the three-state model using the rate constants estimated from the spectra at 2 µM CCh are also shown. Bb, dose–activation relationship. The integrals of the NSCCL currents were numerically calculated. The values obtained are plotted () against the agonist concentration after normalization to the value for the response of each cell to 2 µM CCh (•). The continuous line is a least-squares fit of the Hill function (eqn (14)) with max= 5.9, K= 8.4 ± 0.5 µM and h= 1.1 ± 0.1 (total number of measurements, n= 58). Note that the ordinate is scaled in terms of max (=()). Dashed line is the dose–activation curve predicted from the results of the spectral analysis based on the three-state model. See text for further explanation.

Figure 4Ba summarizes the results of our experiments in which we examined the effects of changing the agonist concentration, c, on the noise spectra of the CCh-evoked NSCCL currents. As can be seen, the values of f₁, f₂ and S₁(0)/S₂(0) did not change dramatically in the range 0.5 µM < c < 10 µM. Consequently, the spectrum of NSCCL currents tended to be shifted upward without largely changing its shape with increasing agonist concentration in this range (compare the spectra at 0.5 and 2 µM CCh in Fig. 4A). Although two Lorentzians were still required to fit the five spectra for the NSCCL current evoked by 20 µM CCh, the two-component nature was obscured at this agonist concentration where the difference between f₁ (50 ± 12 Hz) and f₂ (98 ± 13 Hz) became narrower and S₁(0)/S₂(0) reduced to 2.5 (spectra not shown). In the range 50 µM < c < 200 µM, the spectra were well fitted with the single Lorentzian function with corner frequencies of 75–85 Hz, and no second Lorentzian component was clearly resolved in any of the 15 spectra for the NSCCL currents evoked by CCh in this concentration range (see Fig. 4A and 100 µM CCh). Figure 4Ba includes the graphs of f₁, f₂ (continuous lines) and S₁(0)/S₂(0) (dashed line) calculated based on the three-state model (Scheme 1) using the rate constants given above. These graphs appear to describe fairly well the observed changes of the spectral parameters caused by changing the agonist concentration (Fig. 4Ba). Based on the three-state model, the disappearance of the second Lorentzian component in the range c > 50 µM can mainly be attributed to the steep increase of S₁(0)/S₂(0) in this range (Fig. 4Ba, dashed line), which corresponds to the reduction or vanishing of the A + R state caused by high concentrations of the agonist occupying R.

Figure 4Bb shows the dose–activation relationship for the CCh-evoked NSCCL currents which were used for the spectral analysis described above. The data were fitted with the Hill function with K= 8.4 ± 0.5 µM and h= 1.04 ± 0.06 (total number of measurements, n= 58). It is worth noting that the K value is close to the value (6.6 µM) calculated by eqn (12) using the rate constants obtained from the spectra at 2 µM (see above).

Unlike the NSCCL currents, pure NSCCS currents or the NSCCS component of CCh-evoked currents tended to become smaller when stimulation by CCh was repeated. This tendency was especially marked if the agonist concentration exceeded 10 µM. However, an essentially similar dose–activation relationship (K= 9.3 ± 0.7 and h= 1.07 ± 0.08; n= 128) was obtained in the range 0.04–10 µM CCh if responses containing a NSCCS component in various ratios (0 < |µ_s|/|µ|_max < 0.8 at 2 µM CCh) were included. This is consistent with the idea that CCh activates NSCCL and NSCCS through the same type of muscarinic receptor.

Using the values of the rate constants, the equilibrium probabilities of the open state of NSCCL (Scheme 1) at a given agonist concentration can also be estimated using the set of equations labelled eqn (10). At 2 µM CCh, for example, we have p_AR*= 0.08, p_AR= 0.16 and p_A+R= 0.76. The smallness of the value of p_AR* is consistent with the fact that the µ–² relationships for the NSCCL currents gave a linear appearance at this agonist concentration (Fig. 2Ba; see also eqn (3)).

Spectra with two Lorentzian components very similar to those obtained for the responses purely attributable to the opening of NSCCL alone were also obtained from many responses which gave µ–² plots with two components. This was not unexpected because even relatively small NSCCL currents gave spectral densities of at least an order of magnitude larger than the largest NSCCS currents over the entire frequency range where comparison could be made.

NSCCS.  Spectral analysis did not resolve any clearly defined Lorentzian components in many of the cells which gave pure NSCCS currents in response to CCh (2 µM) in normal PSS at the holding potential of –50 mV. This was probably due to the limited frequency resolution as a result of the very low amplitude of the noise. Increases in noise were more clearly observed when Ba²⁺ was used as the charge carrier for NSCCS (see Fig. 7A). Figure 5 shows the spectrum of a NSCCS current evoked by 2 µM CCh during superfusion with a solution containing Ba²⁺ (92 mM) as the only significant cation producing an inwardly directed electrochemical gradient. It appeared evident that the spectrum had more than one Lorentzian component, even though the resolution at frequencies above 100 Hz was still limited (Fig. 5). By fitting the spectral data with the sum of two Lorentzians, we obtained the following estimates: f₁= 3.1 Hz, S₁(0) = 0.063 pA² s, f₂= 152 Hz and S₂(0) = 0.00071 pA² s.

View larger version (17K): [in this window] [in a new window]   Figure 5.  Power spectrum of NSCCS current Power spectrum of the noise of the CCh-evoked NSCCS current such as the one shown in Fig. 7. CCh (2 µM) was applied during superfusion with a solution containing Ba2+ (92 mM) as the only significant cationic charge carrier (see the lower trace of Fig. 7A). This net spectrum was calculated essentially as described in Fig. 4A, except that the segment size was set at 8192 samples for better frequency resolution. Forty-eight control spectra and 18 spectra during the application of CCh were averaged in this case. Only the part of the symmetrical two-sided net spectra within the frequency range 0.4–600 Hz is shown. The double Lorentzian function was fitted to the data in this range by non-linear least-squares method. The corner frequencies (f1 and f2) of the Lorentzian components (dashed lines) are indicated by arrows. See text for further explanation.

Relative cation selectivity of NSCCL and NSCCS

Figure 6 shows typical results of the cation-substitution experiments that we carried out in order to examine the relative cation selectivity of NSCCL. For this purpose, we chose relatively rare cells that gave a current purely attributable to NSCCL openings in response to an initial trial application of CCh (2 µM) in PSS at a holding potential of –50 mV (Fig. 6A, top; see also Fig. 2Aa and Ba). CCh (2 µM) was then applied to the cells at various holding potentials during superfusion with a solution containing an alkaline metal ion (Li⁺, Na⁺ or Cs⁺; 138 mM) or an alkaline earth metal ion (Mg²⁺, Ca²⁺, Sr²⁺ or Ba²⁺; 92 mM) as the only significant cationic charge carrier. From this type of experiment it was readily apparent that the alkaline metals gave a larger current than did the alkaline earths (see Fig. 6A). To make a quantitative comparison, we estimated the unitary current amplitude at each holding potential either by non-stationary variance analysis or by direct measurement of the size of current steps. As shown in Fig. 6B, the E_h–I relationships obtained differed not only in the slope but also in the reversal potential E_r, indicating a difference in the permeability of NSCCL to the ions. Table 1 gives the values of E_r for the substitute cations (X) together with the permeability ratios P_X/P_Na calculated using eqn (13). We see from Table 1 that the cations fall in the permeability sequence Cs⁺ > Na⁺ > Li⁺ > Ba²⁺ Sr²⁺ > Ca²⁺ > Mg²⁺. NSCCL thus exhibited a mild selectivity in favour of the monovalent cations, although it was also measurably permeable to the divalent cations including Ca²⁺.

View larger version (15K): [in this window] [in a new window]   Figure 6.  Effects of total substitution of the extracellular cations on NSCCL current Na+, K+, Ca2+ or Mg2+ ions in the bath solution were totally replaced by alkaline metal ions (138 mM) or alkaline earth metal ions (92 mM). Whole-cell current was evoked under voltage clamp by the application of CCh (2 µM) in ciliary myocytes which gave a current mostly attributable to openings of NSCCL, as judged by non-stationary variance analysis. A, typical responses obtained from a cell at a holding potential (Eh) of –50 mV. The cell was first equilibrated in normal PPS and CCh (2 µM) was applied (uppermost trace). The µ–2 relationship of this response was well fitted by a line with a slope of 1.7 pA (not shown). CCh was then applied to the same cell during superfusion with a solution containing only Ca2+, Ba2+ or Cs+ as the charge carrier. To minimize damage to the cell, the cell was rested in normal PSS for 3–5 min before each cation exchange (not shown). CCh evoked a slightly deteriorated but still clear response when applied in PSS at the end of the experiment (compare with the response at the start). B, the dependence of the unitary NSCCL current on Eh. In experiments similar to those shown in A, CCh (2 µM) was applied at various potentials during superfusion with a solution containing only Ca2+ (), Ba2+ (), Na+ ( and ) or Cs+ ( and ) as the charge carrier. The unitary currents were estimated either by non-stationary variance analysis (open symbols) or by direct measurement of the size of the current steps (filled symbols) as for those shown in Fig. 1B. Each symbol represents the average of the values obtained from 3–4 cells. Note that the E–I relationships obtained using the different charge carriers differ not only in the slope but also in the reversal potential (see Table 1).

µ–

On-cell patch clamp recording of the NSCCL current

Although we tried to record single-channel currents of NSCCL from on-cell membrane patches in more than 250 cells, most of the trials were unsuccessful. This was not unexpected because the density of NSCCL that were open during stimulation by CCh is extremely low, as shown above. We were only able to record CCh-evoked NSCCL openings under on-cell membrane-patch voltage clamp in six cells.

Figure 8 shows typical results obtained from one of the successful trials. The pipettes used for these experiments were filled with PSS containing 2 µM CCh and 2 µM iberiotoxin, a specific blocker of BK-type K⁺ channels (Galvez et al. 1990), and their tips were plugged with a small amount of normal PSS containing neither CCh nor iberiotoxin by immersing in PSS and applying a weak negative pressure (–50 to –100 mmHg for 3–10 min) from the other end. The bath was perfused with high (100 mM) K⁺ PSS. After establishment of giga-seal (t= 0 min), the potential in the pipette (E_p) was held constant at 0 mV, and a series of rectangular voltage pulses (–E_p changing from –80 mV to +70 mV at 10–20 mV steps) of 250 ms duration at 5 s intervals was intermittently applied. At the start of the recording (t= 0–5 min; Fig. 8A), the opening of Ca²⁺-dependent K⁺ channels (BK type) carrying large outward current at positive potentials was often observed. Application of 2 µM CCh to the bath during this period caused no discernible change in the channel activity in the membrane patch in any of the cells examined (not shown). The BK channel stopped opening by 15 min and in the six successful trials, single-channel openings started to be observed (Fig. 8B). After the openings had started, they were unaffected by bath application of 10 µM atropine in any of the six trials. The plot of the unitary current amplitude against –E_p (not shown) could be fitted with a straight line which crossed the –E_p axis at a voltage near 0 mV, and from the slope of this regression line, the unitary conductance of 35 pS was estimated, indicating that the single-channel currents were produced by the opening of NSCCL. The BK channel activity and its gradual disappearance, the latter of which is probably due to the arrival of iberiotoxin at the extracellular surface of the membrane patch by diffusion, were observed not only in the six successful trials but also in most of the other unsuccessful trials where no NSCCL openings were observed. (Iberiotoxin (4.3 kDa) is known to inhibit BK channels at subnanomolar concentrations (Galvez et al. 1990), whereas at least 0.1 µM CCh (147.19 Da) is required to activate NSCCL (see Fig. 4Bb). Given such an exceedingly high affinity of iberiotoxin to BK channels, it is not surprising that the effect of iberiotoxin, which has a larger molecular size than CCh, usually appeared earlier than did that of CCh. Despite the relatively large difference in the molecular sizes, the ratio of the diffusion coefficients of CCh and iberiotoxin is estimated to be at most 5, using the well-known rule of thumb that the diffusion coefficient is inversely proportional to the cube root of the molecular mass.) In two of the six cells, the number of open channels gradually increased and reached a steady level by 20–30 min (Fig. 8C), but in the other four cells such an increase did not occur and only a small number of NSCCL openings continued to be observed for more than 60 min (not shown). The reason for this difference is not clear from the present experiments.

View larger version (24K): [in this window] [in a new window]   Figure 8.  Unitary NSCCL openings recorded under on-cell membrane-patch voltage clamp Voltage clamp recordings from an on-cell membrane patch. The pipette used was filled with PSS containing 2 µM CCh and 2 µM iberiotoxin, and its tip was plugged with a small amount of normal PSS containing neither CCh nor iberiotoxin by immersing in PSS and giving suction from the other end. The bath was perfused with high K+ (100 mM) PSS. After establishment of a giga-seal (t= 0 min), the potential in the pipette (Ep) was held constant at 0 mV, and a series of rectangular voltage pulses (–Ep changing from –80 mV to +70 mV in 10–20 mV steps) of 250 ms duration at 5 s intervals was intermittently applied. The responses elicited by the voltage pulse series at 5 min (A), 15 min (B) and 25 min (C) are shown. In the early stage (0–10 min), opening of a Ca2+-dependent K+ channel (BK type) carrying large outward current at positive potentials was often observed (A). As the BK channel stopped opening by 15 min, probably due to the arrival of iberiotoxin at the extracellular surface of the membrane patch by diffusion, single-channel openings of NSCCL began to be observed (B). In this and one other cell, the NSCCL openings gradually increased and reached a steady level by 20–25 min (C). In the other 4 cells where single-channel openings of NSCCL were observed, such an increase did not occur and only an small number of openings continued to be seen for more than 60 min (not shown). See text for further details.

Possible involvement of G-protein-dependent pathway in the muscarinic signal transduction

When we used pipette solutions containing no GTP, as we did in our previous experiments (Takai et al. 1997), currents accompanied by a clear increase in noise were observed in only 38 out of the 254 cells which gave clear inward currents in response to 2 µM CCh at the holding potential of –50 mV. In contrast, the presence or absence of GTP in pipettes did not change the properties of the NSCCS currents to a considerable degree. For CCh-evoked currents of smooth appearance recorded from 218 cells using a pipette solution containing no GTP, variance analysis estimated a mean value of 4.88 ± 0.06 fA (n= 288 responses), which is not significantly different from the value given above for the NSCCS currents recorded using a pipette solution containing GTP.

As shown in Fig. 9A, replacement of GTP in the pipette solution with its poorly hydrolysable analogue GTPS gradually caused spontaneous opening of NSCCL in the absence of CCh, suggesting a G-protein-linked signalling mechanism. CCh (2 µM), when applied 1–2 min after the establishment of the whole-cell configuration, accelerated the appearance of the effect of GTPS (Fig. 9B). In 9 out of the 11 cells in which similar results were obtained, CCh not only activated NSCCL but also caused a marked change in the mean current level, which was probably caused by opening of NSCCS (Fig. 9B). The effect on the mean current level was reversible, in contrast to that on NSCCL which was sustained after removal of CCh. Essentially the same results were obtained if the concentration of GTPS was increased to 360 µM (observation in 2 cells). These results suggest that the signals from the muscarinic receptors are sent to NSCCL by a G-protein-linked mechanism whereas they reach NSCCS through some different pathway which might conceivably be independent of G-proteins (see Discussion).

View larger version (22K): [in this window] [in a new window]   Figure 9.  Activation of NSCCL by intracellular application of GTPS Whole-cell currents were recorded using pipettes filled with solution containing 180 µM GTPS in place of GTP. The membrane potential was clamped at –50 mV. Aa, 5–10 min after establishment of the whole-cell configuration, spontaneous openings of NSCCL were observed in the absence of CCh. Ab, part of the trace is presented on an expanded time scale, to show unitary channel openings. B, application of CCh (2 µM) accelerated the appearance of the effect of GTPS. CCh not only activated NSCCL but also caused a marked change in the mean current level indicating activation of NSCCS. Note that the effect on the mean current level was reversed, whereas that on NSCCL was sustained, after removal of CCh.

Inhibitory effect of intracellular Ca²⁺ on NSCCS

Figure 10 shows the effects of increasing the intracellular Ca²⁺ concentration [Ca²⁺]_i on the CCh-induced currents. The bath was perfused with normal PSS or a solution in which alkaline and alkaline earth cations in PSS were totally replaced with either Ba²⁺ or Sr²⁺. The pipettes used were filled with solution containing 70 or 400 nM free Ca²⁺. It has been shown in several mammalian cells that elevation of [Ca²⁺]_i activates some types of non-selective cation channel. In the bovine ciliary muscle, however, the current elicited by CCh was suppressed by nearly 80–85% when [Ca²⁺]_i was increased from 70 to 400 nM (Fig. 10; PSS), confirming our previous observations (Takai et al. 1997). Still more marked (90–95%) reduction of the current amplitude was observed when the extracellular cations were totally replaced with Ba²⁺ or Sr²⁺, to which NSCCS exhibits considerably higher permeabilities than it does to monovalent cations (see Table 1 above). In our previous experiments, the opening of NSCCL was clearly observed even when the [Ca²⁺]_i was increased to 700 nM, indicating that NSCCL is much less sensitive to elevation of [Ca²⁺]_i. The reduction of the CCh-induced current caused by increasing [Ca²⁺]_i thus appears to be due mostly to inhibition of NSCCS.

View larger version (11K):