Journal of Physiology (1990), 423, pp. 453-473 With 11 figures Printed in Great Britain

453

THE WHOLE-CELL Ca2+ CHANNEL CURRENT IN SINGLE SMOOTH MUSCLE CELLS OF THE GUINEA-PIG URETER

BY R. J. LANG From the Department of Physiology, Monash University, Clayton, Victoria 3168, Australia

(Received 7 June 1989) SUMMARY

1. Calcium channel currents were recorded in single, enzymatically isolated smooth muscle cells of the guinea-pig ureter using a single-electrode whole-cell voltage clamp technique. Calcium and barium currents through voltage-activated Ca2+ channels were recorded in cells dialysed with Cs+- or Na+-containing saline which suppressed K+ currents. 2. Inward currents in Ca2+ (1P5-75 mM) or Ba2+ (1P5-75 mM) were recorded at potentials positive to -50 to -30 mV. Inward currents were maximal at 0 mV in 1-5 mM-Ca2+ and at + 10 mV in 7-5 mM-Ba2t. Current flow through Ca2+ channels in Cs+-filled cells (in 1-5 mM-Ca2+ or 7-5 mM-Ba2+) changed from inward to outward at potentials positive to + 70 mV. In Na+-filled cells this reversal potential was between + 50 and + 60 mV. 3. Replacing Ca2+ or Ba2+ with Co2+ (1P5 mM) blocked all inward current flow through these Ca2+ channels; outward currents at potentials positive to +40 mV, however, were increased. Cadmium (100 uM) and nifedipine (0-1-10 ftM) reduced both inward and outward current flow. 4. Calcium channel activation showed a sigmoidal relationship with membrane potential; the potential of half-maximal activation was - 8-4 mV in 1-5 mM-Ca2+ and - 10-8 mV in 7-5 mM-Ba2+. The maximum membrane conductance to Ca2+ (in 1-5 mM-Ca2+) was 2-57 nS/cell or approximately 0 05 mS/cm2. 5. Evidence for a voltage-dependent inactivation mechanism included (a) the time-dependent relaxation of the outward currents at potentials positive to the reversal potential and (b) a steady-state inactivation (foo(v)) vs. membrane potential relationship (in 7.5 mM-Ba2+) which ranged between -80 and 0 mV, with a halfmaximal availability at -40 5 mV. 6. The voltage dependencies of the inward current elicited from -80 and -30 mV were similar, suggesting that depolarization activated only L-type Ca2+ channels. 7. It was concluded that the processes controlling the time course of the Ca2+ current in single ureteral cells bathed in physiological concentrations of Ca2+ were mostly voltage-dependent. INTRODUCTION

The presence of a voltage-activated Ca2+ current underlying the action potential in smooth muscle (Brading, Biilbring & Tomita, 1969) is now well established in a MS 7733

454

R. J. LANG

number of whole-tissue (Mironneau, 1973; Vassort, 1975; Inomata & Kao, 1976; Hirst, Silverberg & van Helden, 1986) and single-cell preparations (Walsh & Singer, 1981; Bolton, Lang, Takewaki & Benham, 1985; Kl6ckner & Isenberg, 1985; Mitra & Morad, 1985; Ohya, Terada, Kitamura & Kuriyama, 1986). The amplitude and time course of this Ca2+ current, however, shows a considerable degree of variation between smooth muscles. For example, in single cells of the bladder or ileum of the guinea-pig, rabbit portal vein and from the rat vas deferens or myometrium the Ca2+ current is relatively large (hundreds of picoamperes per cell), with a transient time course, activating and inactivating rapidly so that the current, and consequently the action potential, is mostly complete within several hundred milliseconds (Droogmans & Callewaert, 1986; Amedee, Mironneau & Mironneau, 1987; Nakazawa, Matsuki, Shigenobu & Kasuya, 1987; Nakazawa, Saito & Matsuki, 1988). In single cells of the rabbit ear artery, or guinea-pig cerebral arteriole, the Ca2+ current is generally an order of magnitude smaller. It also shows rapid activation and a complex decay, part of which may last for several seconds (Hirst et al. 1986; Aaronson, Bolton, Lang & MacKenzie, 1988). Explanations for these various time courses of the Ca2+ current in smooth muscle have evoked the presence of at least two populations of Ca2+ channels, with differing kinetics and voltage sensitivities (Bean, Sturek, Puga & Hermsmeyer, 1986; Loirand, Pacaud, Mironneau & Mironneau, 1986; Benham, Hess & Tsien, 1987; Yatani, Seidel, Allen & Brown, 1987; Aaronson et al. 1988; Nakazawa, et al. 1988; Yoshino, Someya, Nishio & Yabu, 1988) and mechanisms of inactivation dependent on the membrane potential and/or the previous entry of Ca2+ (Jmari, Mironneau & Mironneau, 1986; Ganitkevich, Shuba & Smirnov, 1987; Ohya, Kitamura & Kuriyama, 1988). This paper describes whole-cell voltage clamp measurements of the Ca2+ current in single smooth muscle cells isolated from the guinea-pig ureter (Imaizumi, Muraki & Watanabe, 1989; Lang, 1989). Calcium channel currents were recorded at Ca2+ concentrations (1-5 mM) close to physiological and in raised concentrations of Ba2+ (7'5 mM) which gave relatively large Ca2+ channel currents, but which did not lead to a substantial change in their voltage sensitivities (cf. Aaronson et al. 1988). Depolarization evoked a rapidly activating, slowly inactivating Ca2+ current. In contrast to other smooth muscles, inactivation of the Ca2+ current in these ureteral cells at physiological Ca2+ concentrations was little affected by Ca2+ entry, which may well explain, in part, the long-lasting potentials and contractions recorded in the intact tissue, particularly in the presence of excitatory agonists (Shuba, 1979). METHODS

Single smooth muscle cells were separated from the mid-portion of the guinea-pig ureter by a 90 min incubation (at 37 °C) in a physiological saline containing collagenase (0-2 mg/ml; Cooper Biomedical), elastase (1I25 U/ml; Sigma), bovine serum albumin (2 mg/ml; Sigma) and 30 Ca2l (Lang, 1989). Membrane currents were recorded at room temperature (22-25 °C) from single cells with an Axopatch 1B patch clamp apparatus (Axon Instruments) and low-resistance electrodes (2-7 M!Q) (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Cells were initially impaled in a bathing solution containing (mM): NaCl, 126; KC1, 5'9; sodium HEPES, 6; glucose, 11; MgCl2, 1'2 and CaCl2, 1-5; adjusted to pH 7-4 with 5 M-NaOH. Currents through Ca2+ channels were recorded in a similar saline in which K+ was substituted by Cs+, tetraethylammonium (TEA, 5 mM) added and Ca2+ or Ba2+ varied between 1'5 and 7'5 mM; pH was

/tM-

Ca2' CHANNEL CURRENTS IN SINGLE URETER CELLS

455

adjusted with 5 M-TEA-OH. The pipette solution contained (mM): sodium HEPES, 6; ATP, 3; EGTA, 3; MgCl2, 2-9; glucose, 11; TEA, 5; CsCl or NaCl, 130 as indicated and buffered to pH 7-4 with TEA-OH (Lang, 1989). Data were recorded on an FM tape-recorder (Tanberg) and then analysed after digitization with a Labmaster analog-to-digital interface using an Arrow-AT personal computer and p-CLAMP software (Axon Instruments). The time course of the decay of Ca2+ channel currents was often fitted with a series of exponentials of the form:

F(t) = AO +A1 exp (t/Tl) +A2 exp (t/T2) + The amplitude of the constants, AO ...A. was determined by a multiple linear-regression analysis,

the time constants T1 ... T. were calculated using an iterative simplex algorithm that minimized the residual error obtained from the regression calculation. On the other hand, the time course of the rising phase of these inward currents was fitted in a similar manner to a single exponential raised to a power (n) of the form:

F(t) = AO +A1( 1-exp (tlTl))n. RESULTS

Time-dependent membrane currents could be recorded at potentials positive to -50 mV in most Cs+-filled ureteral cells in Ca2+- or Ba2+-containing (1P5-7-5 mM) saline when triggered by depolarizing command pulses (0 4-1 0 s duration), from a holding potential of -80 mV (Fig. IA). In Fig. IA the inward current at 0 mV in 1-5 mM-Ca2+ (ICa) increased approximately twofold when the extracellular Ca2+ concentration was raised to 4-5 mm, and only slightly further in 7-5 mM-Ca2+. The amplitude of the inward current in 1-5 mM-Ba2+ ('Ba) was similar to that in 1-5 mMCa2+ and increased approximately twofold and threefold in 4-5 and 7-5 mM-Ba2+

respectively (Fig. LB). Inactivation of Ca2+ channel currents has been demonstrated to be dependent on both the membrane potential (Jmari et al. 1986) and on the previous entry of Ca2+ in a number of excitable cells (Ashcroft & Stanfield, 1982; Eckert & Chad, 1984). In smooth muscle, a slowing down of inactivation when the external Ca2+ is exchanged for Ba2+ has been reported in the taenia coli (Ganitkevich et al. 1987) and small intestine of the guinea-pig (Droogmans & Callewaert, 1986) and rat myometrial cells under culture (Amedee et al. 1987). A U-shaped dependence of both the rate and extent of inactivation on potential, such that maximal inactivation occurred near the peak of the ICa current-voltage curve, has also been demonstrated in the guineapig bladder (Kl6ckner & Isenberg, 1985) and rat myometrium (Jmari et al. 1986). In Fig. 1 C the inward currents at 0 mV in 1-5 mM-Ca2+ and 1-5 mM-Ba2+ have been superimposed, after subtraction of their background membrane and capacitive currents, recorded in 1.5 mMCo2+. This cation replacement had little affect on the rate of inactivation during these 1 s depolarizations. Increasing the external Ca2+ usually accelerates inactivation of ICa in smooth muscle (Jmari et al. 1986; Ganitkevich et al. 1987). In the present experiments ICa was not accelerated when the Ca2+ concentration was raised to 7-5 mm (Fig. 1 C). These results suggest that if a mechanism of inactivation dependent on the previous entry of Ca2+ is present in ureteral cells, it contributes only slightly to the time course of ICa in ureteral cells (see below).

R. J. LANSG

456

Ureteral cells were generally studied in 1-5 mM-Ca2+ and 7-5 mM-Ba2+, under divalent cation concentrations which are physiologically relevant or where large inward currents are measured routinely. In Fig. 2A, membrane currents (in 1-5 mmCa2+ and 7X5 mM-Ba2+) have been evoked at the potentials indicated between -50 A

Ca2+ (mM) 1.5 mM-Co2+

B5rnBa2+ m

J100 pA

7

m -Ca12MC

mm-Ca72

15 mm-Ba52

100 Ms

C 1.5

7 m-a2 |

100 pA

100 ms

Fig. 1. Inward Ca2+ channel currents recorded in a single Cs+-filled ureteral cell in Ca2+_ containing (A) and Ba2+-containing (B) solutions; depolarizations to 0 mV from a holding potential of -80 mV. Background membrane and capacitive currents at 0 mV revealed by replacing Ca2+ or Ba2+ with 1-5 mMCo2+. C, superimposed inward currents in Ca2+ and Ba2+ (1 5 and 7 5 mM) after previous subtraction of background currents.

and + 100 mV and superimposed. Threshold in some cells was near -50 mV, in other cells it was closer to -40 mV. Substantial activation of an inward current always occurred at -30 mV. These inward currents in 1-5 mM-Ca2+ or 7-5 mM-Ba2+ showed little inactivation near threshold. Currents ICa and IBa were maximal and inactivated most rapidly near 0 and + 10 mV respectively. At potentials positive to + 10 mV the membrane current in Ca2+ or Ba2+ changed progressively from a decaying inward current to a decaying outward current. Near + 70 mV no time-dependent currents were recorded. In Fig. 2B the early peak current ('peak) and the current at the end of these 400 ms depolarizations (I400) in 1-5 mM-Ca2+ (open symbols) and 7 5 mM-Ba2+ (filled symbols)

r.O~

Ca2& CHANNEL CURRENTS IN SINGLE URETER CELLS

457

are plotted against the membrane potential. It can be seen that Ipeak and '400 intersected at + 70 mV in both Ca2+ (1P5 mM) and Ba2+ (7-5 mm) saline, at the same null potential where no active membrane current was recorded upon depolarization.

Effects of low-Na+ solution It has been suggested previously that the inward current underlying the plateau of the action potential, recorded extracellularly in whole strips of guinea-pig ureter, A 1.5 mM-Ca i

A

7.5 mM-Ba

1-o

50

mV

-40

-30 -20

-10

r

50 pA 100

ms

M. '

200 1

0;Lr

-200

Ba2+

1-5 mM 7.5 mM

/Peak /400

.4

100 80 70 60 40

Ca2+

B

0-0 A-&

0

" *--

+010--

/

V

-400 K .4. 0 40 80 -120 -80 -40 Membrane potential (mV)

20

10

Fig. 2. A representative family of Ca2+ channel currents recorded from a Cs+-filled cell in 1-5 mM-Ca2+ (left column) and 7-5 mM-Ba2+ (right column) at the potentials indicated; holding potential -80 mV. B, plot of current-voltage relationship in 1-5 mM-Ca2+ (peak inward current, Speaks 0; current at end of these 400 ms voltage steps, I400, A) and in 7.5 mM-Ba2+ ('peak' 0; A). Note that an inward current is activated at potentials positive to-40 mV; peaks at 0 mV in 15 mm-Ca2+, +10 mV in 7*5 mM-Ba2+ and becomes a decaying outward current at potentials positive to + 70 mV. '400,

arises from the flow of both Ca2+ and Na+ ions (Shuba, 1979). Recently, the action potential duration and the late portion of ICa in single ureteral cells were both reduced when most of the external Na+ was removed. (Imaizumi et al. 1989). Removal of external Ca2+ in the presence of normal Na+, however, completely abolishes ICa in single ureteral cells (Imaizumi et al. 1989; Lang, 1989), perhaps

R. J. LANG

458

suggesting that the Na+ component of the inward current may be activated only upon Ca2+ entry, i.e. Ca2+-activated Na+ (or non-selective cationic) channels or an electrogenic Na±-Ca2+ exchange mechanism are being activated. The movement of Na+ ions through Ca2+ channels can, however, be tested by

l

l

132 mM-Na+

> I. -02

mm-Na' ~~~~~~~~~~12

-_60

d100 pA

and

200 ms

Fig. 3. Effects of removing approximately 90% of the external Na+

on Ca2+ channel currents in 7-5 mM-Ba2+. Superimposed family of currents every 20 mV between-60 and +100 mV in Ba2+, 132 mM-Na+ (left panel) and Ba2+, 12 mM-Na+ (right panel); Na+ replaced with TEA, holding potential -80 mV.

using Ba2+ instead of Ca2+ as the charge carrier, which should prevent any internal Ca2+-activated mechanisms. In Fig. 3, IBa (7 5 mM-Ba2+) has been recorded in saline containing normal Na+ (132 mM) and in approximately one-tenth of its normal concentration (12 mM), Na+ being replaced by an equimolar concentration of TEA. Membrane currents between -60 and + 100 mV were elicited by depolarizing steps, increasing 20 mV in amplitude. There was no consistent change in the amplitude or time course Of IBa. For example, in some cells Na+ replacement led to a slight increase in the amplitude of IBa, presumably due to a greater block of residual background currents. In other cells, IBa was slightly reduced. However, when these cells were returned to normal Na+, IBa remained smaller because of time-dependent run-down of these Ca2+ channel currents. These results suggest that Ca2+ is the main permeant ion through ureteral Ca2+ channels in normal physiological saline. It is well known, however, that Na+ ions can flow through Ca2+ channels when the external Ca2+ concentration is fixed artificially at low levels ( 0 99) with time constants T1 = 124 ms and T2 = 695 ms. At -100, -50 and -30 mV, T1 was 77, 159 and 97 ms respectively, while T2 was 366, 602 and 559 ms respectively (cf. Lang, Aaronson, Bolton & MacKenzie, 1987). These results suggests that ureteral cells are unlikely to possess many of the channels responsible for the low-threshold, rapidly-inactivating Ca2+ current reported in some, but not all, smooth muscle so far examined (Bean et al. 1986; Loirand et al. 1986; Benham et al. 1987; Yatani et al. 1987; Aaronson et al. 1988 compared with Nakazawa et al. 1988), unless they have a virtually identical voltage sensitivity to the slowly inactivating, nifedipine-sensitive (see below) Ca2+ channel currents recorded.

Steady-state inactivation of Ca2+ channel currents Changes in the holding potential, from a control potential of -80 mV, altered the amplitude of 'Ba in both a time- and voltage-dependent manner. For example, shifting the membrane potential from -80 to -60 mV reduced 'Ba approximately 15-20 % over 1-2 min; a shift from -80 to -20 mV reduced 'Ba approximately 80 % within 30 s (Fig. 8C). Depolarization also accelerated current run-down such that repolarization to -80 mV seldom restored 'Ba completely to its previous amplitude. The lack of restoration of current amplitude upon repolarization was more pronounced when in 1*5 mM-Ca2+. Steady-state inactivation was therefore calculated only in Ba2+-containing saline and assumed to reflect the voltage dependence of only one population of Ca2+ channels. Cells were held at various potentials for a standard 2 min and stimulated every 30 s. Calcium current run-down was taken into account by repolarizing to -80 mV (for 2 min) between each test holding potential to obtain a new control 'Ba (Fig. 8 C). In Fig. 8D the influence of the holding potential on the availability Of hBa in five cells is illustrated. Current Ipeak at each holding potential was expressed as a fraction of its preceding Ipeak at -80 mV and plotted against potential. The sigmoidal, steadystate inactivation (or availability) curve (fo(v)) was fitted through the data, by a least-squares fit, with

f(v) = {1 + exp [(V-VV.5)/k]})' with a potential of half-maximal availability (V0.5) of -40 5 mV and a slope, k, of 13-1 mV. This curve indicates that at a resting membrane potential of -60 mV (as measured with intracellular microelectrodes (Kuriyama, Osa & Toida, 1967) about 80 % of the total population of Ca2+ channels would be available for activation and that the use of the holding potential, -80 mV, in the present experiments increased this availability to nearly 100 %. In Fig. 8D the steady-state activation curve for IBa (Fig. 6D) is also plotted. Between -40 and 0 mV there is a considerable overlap of these two curves, suggesting that there is a membrane potential 'window' at which a percentage (< 10-15 %) of these Ca2+ channel currents do not inactivate (Kl6ckner & Isenberg, 1985; Droogmans & Callewaert, 1986). This non-inactivating current window suggests that ICa may well be responsible for the plateau of the ureteral action

Ca2+ CHANNEL CURRENTS IN SINGLE URETER CELLS A

c -80 mV

-60

-40

-50

465

-20

_iv

1 -0 '

,-

x

B

_Eo 0-5

-8OmV 1 peak

50

-

'

LA-

'is

0/l

A1- /1 z

*-

-1

A,

0.0

-50 /(pA) -100

0

-I 0 40 -40 80 Membrane potential (mV)

-150 *-80

v

-30 mV Difdifference *.e 1-0 *-A 0OA-

/is

H

0

I(pA)

l-

0

0.8

0.2 0.0

-100 '

80

15

0.4-

-A

-50

-150

10 Time (min)

0.6

/peak

50

5

D 1.*0

Iw

-100 -80 -60 -40 -20 0 20 Membrane potential (mV) 0 40 -40 Membrane potential (mV)

80

Fig. 8. Effect of membrane potential on inactivation of Ca2+ channel currents. A, superimposed IBa (7 5 mM-Ba2+) at 0 mV activated from the potentials indicated. B, plot of 's A) triggered from -80 mV (upper panel) current-voltage relationship Of IBa ('peak,'; I, and -30 mV (lower panel). The difference between these currents from the two holding potentials is also plotted in the lower panel (0O A). C, plot Of 'peak of IBa, expressed as a fraction of the first elicited, plotted against time. Note that repolarization to -80 mV after holding the membrane potential at some positive level did not usually restore 'peak to its previous level. D, plot of steady-state inactivation properties of IB4 in five cells in 7-5 mm-Ba2+. The smooth curve through the data represents a Boltzmann equation, fitted by least squares, with a potential of half-inactivation, or availability, of -40 5 mV and a slope factor of 13-1 mV, such that

fcJ,(v) = {1 +exp [(V+0 0405)/0 0131]}'. The steady-state activation curve in 7-5 mM-Ba2+ (from Fig. 7) is also plotted and shows a considerable overlap between -40 and 0 mV, suggesting a range of membrane potential over which a sizeable fraction of these Ca2+ channels do not inactivate.

potential and for the sustained contractures recorded in the presence of excitatory agonists or exposure to raised K± concentrations (Shuba, 1979; Imaizumi et al. 1989).

Effects of nifedipine The dihydropyridine Ca2+ antagonist, nifedipine (0-01-3,UM), caused a dosedependent inhibition of both the inward and outward flow of current through

R. J. LANG

466

ureteral Ca2+ channels (Fig. 9). The blockade of Ipeak and I400 Of IBa (7-5 mM-Ba2+) was equal at all potentials in 04 /lM-nifedipine (Fig. 9B). However, 1 /UM-nifedipine blocked Ipeak more effectively at potentials positive to 0 mV, such that the peak of its current-voltage relationship shifted 10 mV in the negative direction. I400 was A

B



-30

-40

my

-80

ms -30

mV

a

0.8 0.6-

A

d

0.1 pm-nifedipine

0-4 0.2

A-

0

1

2

3

4 14 15 Time (min)

16

17

18

19

Fig. 10. Effects of membrane potential on the inhibitory action of 01 /M-nifedipine. A, IBa at 0 mV (7 5 mM-Ba21) triggered from -80 (a, d) -40 (b, e) and -30 (c, f) mV in the absence (left panel) and presence of 0-1jIM-nifedipine (right panel). B, plot of Ipeak (0) and '400 (A), expressed as a fraction of the first response, against time in the absence and presence of 0 1

/LM-nifedipine.

rapidly inactivating inward current remained. In Fig. 11 inward currents in the presence of 1-5 mM-Ca2+ or 15 mMCo2+ (at the potentials indicated) are compared on an expanded time base. The current-voltage relationship of this transient inward current in Co2+ peaked in amplitude approximately 10 mV negative of the peak amplitude of ICa (Fig. bIB). This transient current was also not affected if nifedipine (1 JtM) or Cd2+ (100 /tM) was added to the bathing solution. An inward Na+ current with similar kinetics and current-voltage relationship has been described recently in single smooth muscle cells of the rabbit main pulmonary

468

R. J. LANG

artery (Okabe, Kitamura & Kuriyama, 1988) and in cultured smooth muscle cells from vascular tissue of neonatal rat (Sturek & Hermsmeyer, 1986). In the pulmonary artery this Na+ current was reduced when the extracellular Na+ concentration was lowered and by low concentrations (> 1 nM) of tetrodotoxin (Okabe et al. 1988); the A

1-5 mM-Co2+

1.5 mM-Ca 2+

-40 mV -30 -20 0 50 pA

50 ms B

Ca

50

50 -

2+

CO+ Co

1.5 mM 'peak *-@

0-0

-

Hi-50 -TI ^ 7 1400j

A-A A-&

0

50~~~~~~~~~~

-100 -

-150 -80

-40 0 Membrane potential (mV)

40

Fig. 11. Evidence of a transient Co2+-insensitive inward current in single ureteral cells. A, superimposed currents in 1-5 mM-Ca2+ (left panel) and 1.5 mM-Co2+ (right panel) at the potentials indicated, displayed on an expanded time base; holding potential -80 mV. B, plot of current-voltage relationship of inward currents in 1-5 mM-Ca2+ ('peak' *; 1400, A) and in 1-5 mM-Co2+ ('peak' 0 ; I400, A).

Na+ current in the cultured vascular cells was relatively less sensitive to tetrodotoxin (60 yUm; Sturek & Hermsmeyer, 1986). It seems likely that the Co2+-insensitive current recorded in ureteral cells arises from the activation of a similar population of Na+ channels and has not been studied further. Analysis of the steady-state inactivation properties of this current in the pulmonary artery, however, showed that it was approximately 70% inactivated at -60 mV (Okabe et al. 1988). If this was the case in single ureteral cells, this transient Na+ current would contribute little to the rising phase of the action potential triggered upon depolarization.

Ca2+ CHANNEL CURRENTS IN SINGLE URETER CELLS

469

DISCUSSION

The internal dialysis of Cs+- or Na+-containing saline into single ureteral cells has allowed an investigation of the currents flowing through voltage-activated Ca2+ channels over a wide potential range (-50 to + 100 mV), with little interference from other time-dependent K+ currents reported previously (Imaizumi et al. 1989; Lang, 1989). Substantial activation of Ca2+ channels occurred at potentials positive to -40 mV, maximal activation was at +20 mV. At more positive potentials ICa changed from a slowly inactivating inward current to a slowly inactivating outward current, such that a genuine reversal potential was recorded near + 70 mV in Cs+filled cells and near + 55 mV in Na+-filled cells. Once fully activated, however, the current-voltage relationship for ICa was not usually linear (i.e. ohmic). There was an inflexion at the reversal potential, suggesting an increase in the Ca2+ channel conductance at more positive potentials (Figs 2, 4, 5 and 10). Similar inflections have been reported in single ventricular (Lee & Tsien, 1984) and adrenal chromaffin cells (Fenwick, Marty & Neher, 1982). The potential range of Ca2+ channel activation in the guinea-pig taenia coli or bullfrog atrial myocytes shifts 10 mV in the negative direction when Ca2+ is replaced by an equimolar concentration of Ba2+ (0-5-9 mM). Conversely, increasing the divalent cation concentration (from 2-5 to 7-5 or 9 mM) produces a similar movement of the activation range in the positive direction (Campbell, Giles & Shibata, 1988a; Ganitkevich, Shuba & Smirnov, 1988). These observations have been explained in terms of a specific binding of divalent cations to negatively charged groups close to the outer surface of the Ca2+ channels. The neutralization of these charges by cation binding is thought to modify the electric field near the Ca2+ channels and therefore the voltage they experience. The different voltage sensitivities between Ca2+ and Ba2+ arises presumably from their differing affinities for these negative binding sites. In ureteral cells it follows that the Ca2+ channel currents in 1i5 mM-Ca2+ and 7.5 mmBa2+ should have nearly identical activation ranges and this was found to be so, as V0.5 = -8-4 mV in 1V5 mM-Ca2+ and = - 10-4 mV in 7-5 mM-Ba2+ (Fig. 7 C and D). Barium, 7-5 mm, has therefore been chosen as the standard with which Ca2+ channels were studied in these ureteral cells. The large shifts of the activation range of IBa in the positive direction, associated with higher Ba2+ concentrations (Aaronson et al. 1988) make extrapolations of Ca2+ channel behaviour under physiological conditions difficult and therefore have not been attempted in the present experiments. The ICa in 7-5 mm-Ca2+ was only slightly larger than that in 4-5 mm-Ca2+ (Figs 1 and 9). Ureteral Ca2+ channels therefore saturate over a similar Ca2+ concentration range as calcium channels in single cells of the guinea-pig taenia coli (Ganitkevich et al. 1988) and cardiac tissue (Tsien, Hess, McCleskey & Rosenberg, 1987; Hagiwara, Irisawa & Kameyama, 1988). IBa did not saturate over this concentration range (1-5-7-5 mM-Ba2+) in common with Ba2+ currents through other Ca2+ channels. These results have been explained previously by postulating the presence of a saturable binding site within, but close to the outer surface of, the Ca2+ channel to which cations must bind before entering the Ca2+ channel. Barium is thought to bind less strongly and, therefore, permeates through the channel with a greater conductance (Tsien et al. 1987; Ganitkevich et al. 1988; Campbell et al. 1988a). %

470

R. J. LANG

It seems likely that Cd2+ and Co2" are binding to this same saturable Ca2l-binding site to block current flow, but with different affinities depending on the nature of the competing permeate ion. For example, replacing the external Ca2+ with Co2+ blocked the inward flow of Ca2+ or Ba2+ currents but did not prevent the outward movement of Na+, or Cs+, at potentials positive to the reversal potential (Fig. 5). This suggests that Co2+ is binding only weakly to the binding site as Na+ or Cs+ are capable of expelling it at positive potentials to allow outward current flow. Cadmium on the other hand binds relatively strongly to this binding site, preventing both the inward and outward flow of current at concentrations tenfold lower than Co2+ (Fig. 6). Competition of Cd2+ with Ca2+ or Ba2+ for the same site, to which Ca2+ has a greater affinity than Ba2 , also explains the greater block of IBa by Cd2 , compared to its blockade of ICa in cardiac ventricular cells (Lee & Tsien, 1983) and single cells of the guinea-pig taenia coli (R. J. Lang & R. J. Paul, unpublished observations). The increase in Ca2+ channel conductance, i.e. the inflexion of the current-voltage relationship, at potentials positive to the reversal potential (Figs 2, 4, 5 and 10), can be explained if a second binding site in the Ca2+ channel is postulated. This second binding site with a much higher affinity for Ca2+ has been invoked to explain the blockade of large inward Na+ currents through Ca2+ channels, recorded in the absence of Ca2+, by concentrations of Ca2+ three to four orders of magnitude smaller than the dissociation constant of the saturable binding site (Tsien et al. 1987). Ureteral cells were always perfused with 3 mM-EGTA so that the internal Ca2+ concentration would be extremely low. The outward flow of Na+ or Cs+ through these channels may well be expected to be larger, particularly when Ca2+ was replaced by

Co2+ (Fig. 5). It is not yet clear by which means inactivation Of ICa in smooth muscle is controlled by the previous entry of Ca2+. Inactivation of ICa, as measured from its decay or from its reduction induced by a preceding depolarization, is directly dependent on Ca2+ influx, increasing when the external Ca2+ is raised or near the peak of its current-voltage relationship (Ganitkevich et al. 1987; Ohya et al. 1988). Decreasing

the internal EGTA concentration also reduces the amplitude of ICa and its inactivation, as measured with a twin-pulse protocol, but does not change dramatically its initial rate of decay or its steady-state inactivation curve (Kl6ckner & Isenberg, 1985; Ohya et al. 1988). Replacing Ca2+ with Ba2+, however, slows the decay of the inward current and removes the dependence of current density from inactivation. These results suggest that a Ca2+-binding site responsible for inactivation may well be on the inner cytoplasmic side of the Ca2+ channel, perhaps in a region of limited access so that Ca2+ may reach regionally high concentrations and to which Ba2+ has a lower affinity. In contrast to other smooth muscles, increasing the external Ca2+ concentration did not lead to any substantial acceleration of ICa in ureteral cells; the replacement of Ca2+ with Ba2+ also had little affect on the decay of the Ca2+ channel current (Fig. 1). This may indicate that (1) the current-dependent component of inactivation of ICa, if present in ureteral cells, is relatively not well developed, (2) this Ca2+-binding site may be already saturated in 1-5 mM-Ca2+ or (3) it has a relatively low affinity for Ca2+, similar to that for Ba2+. Alternately, it should also be pointed out that the amplitude of the Ca2+ conductance (q*ca) in ureteral cells is relatively low

Ca2+ CHANiVNVEL CURRENTS IN SINGLE URETER CELLS

471

(0 05 mS/cm2 in 1-5 mM-Ca21) compared to other visceral smooth muscles, e.g. guinea-pig bladder 0 46 mS/cm2 in 3-6 mM-Ca21 (Kldckner & Isenberg, 1985) so that current-dependent inactivation may not have been triggered substantially. Increasing the external Ca2+ may also fail to further trigger this mechanism of inactivation due to current saturation at concentrations above 4-5 mm (Fig. 1). As a result, the site responsible for any current-dependent inactivation would not 'see' an increased Ca21 influx as the external Ca2+ concentration is raised to 7-5 mm. Saturation of Ca2+ entry and/or the binding site responsible for inactivation has been proposed recently to explain the lack of effect of Ca2+ on the decay of 'Ca in atrial myocytes. When the Ca2+ concentration was raised from 15 to 2-5 mm the decay of ICa was accelerated, but not further when the Ca2+ was raised from 2-5 to 7-5 mm (Campbell, Giles, Hume & Shibata, 1988b). Internal Ca2+, however, may play a role in maintaining the amplitude of ICa in single ureteral cells. Release of Ca2+ from internal stores upon application of caffeine (5 mM) decreased the amplitude of ICa by 10-25 %, without affecting its time course (Imaizumi et al. 1989). These affects were not completely reversible upon the removal of caffeine, perhaps suggesting that internal Ca2+ contributes to Ca2+ channel rundown observed in ureteral cells perfused with low-resistance patch pipettes (Lang, 1989). This work was supported by the NHMRC (Australia) and by the Australian Kidney Foundation. I am grateful to Dr G. D. S. Hirst for his valuable criticism of this manuscript.

REFERENCES

AARONSON, P. I., BOLTON, T. B., LANG, R. J. & MACKENZIE, I. (1988). Calcium currents in single isolated smooth muscle cells from the rabbit ear artery in normal-calcium and high-barium solutions. Journal of Physiology 405, 57-75. AMEDEE, T., MIRONNEAU, T. & MIRONNEAU, J. (1987). The calcium channel current of pregnant rat single myometrial cells in short-term primary culture. Journal of Physiology 392, 253-272. ASHCROFT, F. M. & STANFIELD, P. R. (1982). Calcium inactivation in muscle fibres of the stick insect, Carausius morosus. Journal of Physiology 330, 349-372. BEAN, B. P. (1985). Two kinds of calcium channels in canine atrial cells. Journal of General Physiology 86, 1-30. BEAN, B. P., STUREK, M., PUGA, A. & HERMSMEYER, K. (1986). Calcium channels in muscle cells isolated from rat mesenteric arteries: modulation by dihydropyridine drugs. Circulation Research 59, 229-235. BENHAM, C. D., HESS, P. & TSIEN, R. W. (1987). Two types of calcium channels in single smooth muscle cells from rabbit ear artery studied with whole-cell and single-channel recordings. Circulation Research 61, suppl. 1, 10-1 6. BOLTON, T. B., LANG, R. J., TAKEWAKI, T. & BENHAM, C. D. (1985). Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells. Experientia 41, 887-894. BRADING, A. F., BPLBRING, E. & TOMITA, T. (1969). The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli. Journal of Physiology 200, 637-654. CAMPBELL, D. L., GILES, W. R., HUME, J. R. & SHIBATA, E. F. (1988b). Inactivation of calcium current in bull-frog atrial myocytes. Journal of Physiology 403, 287-315. CAMPBELL, D. L., GILES, W. R. & SHIBATA, E. F. (1988a). Ion transfer characteristics of the calcium current in bull-frog atrial myocytes. Journal of Physiology 403, 239-266. CARBONE, E. & Lux, H. D. (1984). A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurons. Nature 310, 501-502.

472 R. J. LANG DROOGMANS, G. & CALLEWAERT, G. (1986). Ca2+-channel current and its modification by the dihydropyridine agonist Bay K 8644 in isolated smooth muscle cells. Pfluigers Archiv 406, 259-265.

ECKERT, R. & CHAD, J. E. (1984). Inactivation of Ca channels. Progress in Biophysics and Molecular Biology 44, 215-267. FENWICK, E. M., MARTY, A. & NEHER, E. (1982). Sodium and calcium channels in bovine chromaffin cells. Journal of Physiology 331, 599-635. FUKUSHIMA, Y. & HAGIWARA, S. (1985). Currents carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes. Journal of Physiology 358, 255-284. GANITKEVICH, V. YA., SHUBA, M. F. & SMIRNOV, S. V. (1987). Calcium-dependent inactivation of potential-dependent calcium inward current in an isolated guinea-pig smooth muscle cell. Journal of Physiology 392, 431-449. GANITKEVICH, V. YA., SHUBA, M. F. & SMIRNOV, S. V. (1988). Saturation of calcium channels in single isolated smooth muscle cells of guinea-pig taenia caeci. Journal of Physiology 399, 419-436. HAGIWARA, N., IRISAWA, H. & KAMEYAMA, M. (1988). Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. Journal of Physiology 395, 233-253.

HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfluigers Archiv 391, 85-100. HESS, P., LANSMAN, J. B. & TSIEN, R. W. (1986). Calcium channel selectivity for divalent and monovalent cations. Journal of General Physiology 88, 293-319. HESS, P. & TSIEN, R. W. (1984). Mechanism of permeation through calcium channels. Nature 309, 453-456.

HIRST, G. D. S., SILVERBERG, G. D. & VAN HELDEN, D. F. (1986). The action potential and underlying ionic currents in proximal rat middle cerebral arterioles. Journal of Physiology 371, 289-304.

HODGKIN, A. L. & HUXLEY, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology 117, 500-544. IMAIZUMI, Y., MURAKI, K. & WATANABE, M. (1989). Ionic currents in single smooth muscle cells from the ureter of the guinea-pig. Journal ofPhysiology 411, 131-159. INOMATA, H. & KAO, C. Y. (1976). Ionic currents in the guinea-pig taenia coli. Journal ofPhysiology 255,347-378. JMARI, K., MIRONNEAU, C. & MIRONNEAU, J. (1986). Inactivation of calcium channel current in rat uterine smooth muscle: evidence for calcium- and voltage-mediated mechanisms. Journal of

Physiology 380, 111-126. JMARI, K., MIRONNEAU, C. & MIRONNEAU, J. (1987). Selectivity of calcium channels in rat uterine smooth muscle: interactions between sodium, calcium and barium ions. Journal of Physiology

384,247-261. KL6CKNER, U. & ISENBERG, G. (1985). Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig). Pfluigers Archiv 405, 340-348. KURIYAMA, H., OSA, T. & TOIDA, N. (1967). Membrane properties of the smooth muscle of guineapig ureter. Journal of Physiology 191, 225-238. LANG, R. J. (1989). Identification of the major currents in freshly-dispersed single smooth muscle cells of the guinea-pig ureter. Journal of Physiology, 412, 375-395. LANG, R. J., AARONSON, P. I., BOLTON, T. B. & MACKENZIE, I. (1987). Calcium currents in vascular smooth muscle. Proceedings of the Sixth International Symposium on Vascular Neuroeffector Mechanisms 10, 233-240. LEE, K. S. & TSIEN, R. W. (1983). Mechanism of calcium channel blockade by verapamil, D600,

diltiazem and nitrendipine in single dialysed heart cells. Nature 302, 790-794. LEE, K. S. & TSIEN, R. W. (1984). High selectivity of calcium channels in single dialysed heart cells of the guinea-pig. Journal ofPhysiology 354, 253-272. LOIRAND, G., PACAUD, P., MIRONNEAU, C. & MIRONNEAU, J. (1986). Evidence for two distinct

calcium channels in rat vascular smooth muscle cells in short-term primary culture. Pfuigers Archiv 407, 566-568. MIRONNEAU, J. (1973). Excitation-contraction coupling in voltage clamped uterine smooth muscle. Journal of Physiology 233, 127-141.

Ca2+ CHANNEL CURRENTS IN SINGLE URETER CELLS

473

MITRA, R. & MORAD, M. (1985). Ca2+ and Ca2+-activated K+ currents in mammalian gastric smooth muscle cells. Science 229, 269-272. NAKAZAWA, K., MATSUKI, N., SHIGENOBU, K. & KASUYA, Y. (1987). Contractile response and electrophysiological properties in enzymatically dispersed smooth muscle cells of rat vas deferens. PflAgers Archiv 408, 112-119. NAKAZAWA, K., SAITO, H. & MATSUKI, N. (1988). Fast and slowly inactivating components of Cachannel current and their sensitivities to nicardipine in isolated smooth muscle cells from rat vas deferens. Pfiigers Archiv 411, 289-295. OHYA, Y., KITAMURA, K. & KURIYAMA, H. (1988). Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein. Circulation Research 62, 375-383. OHYA, Y., TERADA, K., KITAMURA, K. & KURIYAMA, H. (1986). Membrane currents recorded from a fragment of rabbit intestinal smooth muscle cell. American Journal of Physiology 251, C335-346. OKABE, K., KITAMURA, K. & KURIYAMA, H. (1988). The existence of a highly tetrodotoxin sensitive Na channel in freshly dispersed smooth muscle cells of the rabbit main pulmonary artery. Pfiigers Archiv 411, 423-428. SHUBA, M. F. (1979). Smooth muscle of the ureter: nature of excitation and the mechanisms of action of catecholamines and histamine. In Smooth Muscle: An Assessment of Current Knowledge, chap. 17, ed. BULBRING, E., BRADING, A. F., JONES, A. W. & ToMITA, T., pp. 377-384. Edward Arnold, London. STUREK, M. & HERMSMEYER, K. (1986). Calcium and sodium channels in spontaneously contracting vascular smooth muscle. Science 233, 475-478. TSIEN, R. W., HESS, P., MCCLESKEY, E. W. & ROSENBERG, R. L. (1987). Calcium channels: Mechanisms of selectivity, permeation and block. Annual Review of Biophysics and Biophysical Chemistry 16, 265-290. VASSORT, G. (1975). Voltage-clamp analysis of transmembrane ionic currents in guinea-pig myometrium: evidence for an initial potassium activation by calcium influx. Journal of Physiology 252, 713-734. WALSH, J. V. & SINGER, J. J. (1981). Voltage clamp of single freshly dissociated smooth muscle cells: current-voltage relationships for three currents. Pfiigers Archiv 390, 207-210. YATANI, A., SEIDEL, C. L., ALLEN, J. & BROWN, A. M. (1987). Whole-cell and single channel calcium currents of isolated smooth muscle cells from saphenous vein. Circulation Research 60, 523-533. YOSHINO, M., SOMEYA, T., NiSHIO, A. & YABU, H. (1988). Whole-cell and unitary Ca channel currents in mammalian intestinal smooth muscle cells: evidence for the existence of two types of Ca channels. Pfliigers Archiv 411, 229-231.