Pfliigers Arch (1992) 421:117-124

Journal of Physiology 9 Springer-Verlag 1992

Properties of the inactivating outward current in single smooth muscle cells isolated from the rat anococcygeus I. McFadzean and S. England Pharmacology Group, Division of Biomedical Sciences, King's College London, Manresa Road, London SW3 6LX, U K Received November 12, 1991/Received after revision March 3, 1992/Accepted March 18, 1992

Abstract. The properties of the voltage- and time-depen-

dent outward current in single smooth muscle cells isolated from the rat anococcygeus were studied. The outward current was activated by depolarizations to membrane potentials positive to - 4 0 mV. Activation followed third order kinetics; at + 20 mV, the time for the current to reach half its maximal amplitude was around 55 ms. The current inactivated with a time course that could best be described by a single exponential with a time constant around 1500 ms. The steady-state inactivation curve was voltage dependent over the range - 1 1 0 to - 30 mV, with a half-inactivation point of - 67 mV. Recovery from inactivation followed an exponential time course with a time constant of around 770 ms at - 90 mV. Deactivating tail current analysis revealed that a 10-fold change in the extracellular potassium ion concentration resulted in a 42 mV change in the reversal potential of the current. The current was blocked by 4-aminopyridine, tetraethylammonium, quinine and verapamil with ICs0's - the concentrations producing 50% inhibition of the peak current - of 2 mM, 4 mM, 12 gM and 20 gM respectively. The current was not blocked by Toxin I (100 nM) or glibenclamide (10 gM). The current was still present in cells containing 5 m M EGTA; in these cells, replacing extracellular calcium with cadmium depressed the peak current by around 12 %. This could be explained, at least in part, by a negative shift in the voltage dependence of inactivation. Key words: Transient outward current - Smooth muscle

cells - Patch-clamp - Ion channels - Potassium current

Introduction

Voltage-dependent potassium currents, activated by membrane depolarization have been described in almost Offprint requests to: I. McFadzean

all excitable cell types [14] including smooth muscle cells [18]. Whilst in the past it has been convenient to subdivide these currents into two types, namely, low threshold rapidly activating, rapidly inactivating A-type or transient outward currents and high threshold ( 30 mV), slowly activating, non-inactivating delayed rectifier-type currents, it is becoming clear that this is an over simplification, and that the properties of these currents form a continuum between these two extremes. For example, "classical Hodgkin-Huxley" delayed rectifier currents which inactivate, albeit slowly, have been reported in a number of smooth muscle cell types [5, 15, 19, 24, 26, 30, 32]. Recently we have been working with smooth muscle cells dissociated enzymatieally from the rat anococcygeus, and during the course of these studies we observed that these cells contained such a current. The current appeared to differ to those described in other cell types in that it inactivated over a relatively negative range of membrane potentials, more typical of A-type currents. We decided therefore to study the properties of this current in greater detail, and the results are presented here. A preliminary account of our results has been published in abstract form [23].

Materials and methods Single smooth muscle cells were isolated from the rat anococcygeus using the method described by Amedee and colleagues [2]. Briefly, male Sprague Dawley rats (200--400 g) were killed by cervical dislocation and exsanguination and the anococcygeus muscles removed as described by Giltespie [10]. The muscle was dissected free of surrounding connective tissue and chopped into small pieces. After a 10-min pre-incubation in a physiological satt solution (PSS) at 37~ and containing zero added calcium, the tissue pieces were transferred to a dissociating mixture which consisted of PSS containing bovine serum albumin (5 mg m1-1, fatty acid free), papain (1 mg ml 1), coltagenase (type 1 A; 1.5 mg ml 1), dithioerythritol (2.5 raM) and zero added calcium at 37~ After 50 rain in this solution the tissue was washed twice with enzyme-free solution before being sucked up into a wide-bore Pasteur pipette several times to dissociate the single cells from the muscle. The resultant cell suspension was centrifuged at 1000 rpm for t min and the pellet

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Fig. 1 A - D. The outward current activated by depolarization of a

rat anococcygeus smooth muscle cell. A Outward currents evoked by 3-s step depolarizations to membrane potentials between - 40 mV and + 30 mV from a holding potential of - 80 mV. B The same voltage commands were repeated but from a holding potential of - 30 mV. C The holding potential was returned to - 80 mV, the resuspended in PSS containing 0.75 mM calcium. Droplets of this cell-rich solution were placed in the middle of 35-mm petri dishes and stored in the refrigerator until required for electrophysiological experiments. Membrane currents were recorded using the whole-cell variant of the patch-clamp technique [12]. Current signals were digitized using a TL-I DMA interface (Axon Instruments, Foster City, CA, USA) for "on-line" storage on the hard-disk of a personal computer (Vig 2; Viglen Ltd, London, UK). Data aquisition and analysis was facilitated using "pClamp" software (Axon Instruments). Patch micropipettes contained (mM) NaC1 5, KC1 126, MgC12 1.2, ethylenebis(oxonitrilo)tetraacetate (EGTA) 1, glucose 11, 4-(2hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES) 10, pH 7.2 and had resistances in the range 5 - 7 M~?. The series resistance as measured by whole-cell capacitance neutralisation was typically 10-15Mf2. No series resistance compensation was employed. The largest currents recorded during strong depolarizations were around 600 pA leading to an error in the membrane potential recorded during these currents of, at worst, 9 mV. Currents were leak subtracted using one of two methods. In the majority of experiments, the leak, calculated from the pre-pulse used to remove inactivation of the current, was subtracted from current-records '~ However, in those experiments in which the kinetics of activation of the current were studied, leak subtraction was carried out "on-line" using a P/N protocol in which four hyperpolarising sub-pulses of amplitude a quarter of that of the test pulse were used to calculate the leak. All experiments were performed at room temperature (21 --24~ The extracellular PSS contained (mM) NaC1120, KC16, MgC12 1.2, CaCI2 1.5, Na2HPO4 J .2, glucose 11, HEPES 10, pH 7.2, saturated with oxygen. In experiments in which the extracellular potassium concentration was increased or tetraethylammonium (TEA) was applied in concentrations greater than 5 raM, there was an equimolar replacement of NaC1 with KCI or TEAC1 respectively.

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steps repeated, but in this case the voltage jumps lasted 20 s. D Both peak outward (circles) and steady-state (triangles) current/voltage (I/V) relationships constructed from the raw data in C. The steadystate current was measured at the end of the 20-s jump. Note that the steady-state I/V is linear over the voltage range tested and that the two I/V curves deviate at around - 4 0 mV All enzymes used were obtained from Sigma, Pool9 UK. Drugs used were 4-aminopyridine (4-AP), quinine hydrochloride, TEACI, verapamil hydrochloride and glibenclamide, all from Sigma. Toxin I was a generous gift from Wyeth Research Laboratories, Taplow, UK.

Results

S h o w n in Fig. 1 A is a family of o u t w a r d c u r r e n t s evoked o n d e p o l a r i z a t i o n o f a rat anococcygeus cell f r o m a holding p o t e n t i a l of - 8 0 m V to a range o f c o m m a n d p o t e n tials b e t w e e n - 4 0 m V a n d + 30 mV. Similar c u r r e n t s were seen in all cells tested (n = 39). T h e o u t w a r d currents activated w i t h i n 100 ms o f the onset of the d e p o l a r i z a t i o n (see later) a n d i n a c t i v a t e d partly d u r i n g the course of the 3-s step. I f the same voltage steps were repeated b u t from a h o l d i n g p o t e n t i a l of - 3 0 m V (Fig. 1 B), no o u t w a r d c u r r e n t s were evoked suggesting that the c u r r e n t was fully i n a c t i v a t e d at this h o l d i n g p o t e n t i a l a n d that there was n o n o n - i n a c t i v a t i n g o u t w a r d c u r r e n t activated over this range of c o m m a n d potentials. This is further highlighted in Fig. 1 C where a different cell was held at - 80 m V a n d stepped to the same range of m e m b r a n e potentials b u t in this case for 20 s. T h e o u t w a r d c u r r e n t inactivated completely d u r i n g the longer voltage step. S h o w n in Fig. 1 D are b o t h the peak a n d the steady-state c u r r e n t / voltage ,(I/V) relationships for this cell, the latter measured at the end of the 20-s step. The steady-state I / V relationship is linear between - 80 m V a n d + 30 m V em-

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Fig. 2 A - C. The voltage dependence of activation and inactivation of the outward current. A A family of outward currents evoked at different membrane potentials following a 3-s pre-pulse to - 90 mV. Data such as this was used to construct the activation curve shown in C (open circles) which shows the peak outward current, normalized with respect to the current evoked at a command potential of + 30 mV plotted against command potential. The fitted line was drawn by eye. Each point shows the mean + SEM for data from 5 cells. B A family of currents from the same cell as in A, activated by step depolarizations to a command potential of + 30 mV follow-

ing 3-s pre-putses to a range of membrane potentials. Data similar to this was used to construct the inactivation curve shown in C (filled circles) which shows the peak current, normalized with respect to the current evoked following a pre-pulse to - 1 2 0 mV, plotted against pre-pulse potential. The fitted line is a Boltzman curve of the form I/Im~x = i/{1 +exp[(V--Vh)/Vs]} where Vs, the slope factor, is - 11.8 mV and Vh, the membrane potential for 50% inactivation, is - 6 7 inV. Each point shows the mean _+ SEM for data from 4 ceils

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tivation and inactivation of the outward current. A The activation kinetics of the outward current evoked by a step depolarization from a holding potential of - 70 mV to a command potential of + 20 mV. The solid line is a third order exponential function of the form y = Ao + A i [1 - e x p ( - t/z)]" with a time constant (z) of 16.25 ms, fitted between time zero (the start of the voltage jump) and time = 70 ms. The rate at which the current activated was voltage dependent as shown in B. The natural logarithm of the activation time constant of the third order exponential function is plotted against membrane potential. The fitted line (r = 0.984) shows an e-fold change in the activation time constant every 77 inV. Inactivation of the current could best be described by a single exponential as shown in C, which shows the inactivation of the current at + 20 mV. The solid line is a single exponential function of the form y = Ao + A lexp(-- t/r) with a time constant (r) of 1451 ms, fitted between time = 350 ms and time = 2750 ms

120 phasising the absence of non-inactivating current. The two I / V curves deviate at around - 3 5 m V suggesting that this is the threshold for activation of the outward current. This is shown more clearly in Fig. 2 which shows full activation and inactivation curves for the outward current. The activation curve (Fig. 2 C, open circles) was constructed f r o m experiments such as that illustrated in Fig. 2A. The cell was held at - 1 0 m V - since this seemed to produce the m o s t stable recordings - and was pre-pulsed to - 9 0 mV for 3 s to remove inactivation, before being stepped back to a range of m e m b r a n e potentials between - 7 0 and + 30 inV. The activation curve shows the peak current, normalized with respect to the current evoked at + 30 mV, plotted against m e m b r a n e potential. As mentioned earlier, the current activated positive to - 4 0 mV. The inactivation curve (Fig. 2C, filled circles) was constructed f r o m experiments similar to that illustrated in Fig. 2B. The cell was held at 10 m V and pre-pulsed for 3 s to a range of m e m b r a n e potentials between - 1 0 and - 1 2 0 mV before being stepped to -t-30 mV. The inactivation curve shows the peak current, normalized with respect to the current evoked following a pre-pulse to - 1 2 0 m V , plotted against pre-pulse potential. The fitted line is in the f o r m of a Boltzman equation (see legend, Fig. 2) with a halfinactivation point of - 6 7 m V and slope of - 1 1 . 8 mV. The rate of rise of the current - measured as the time to reach half maximal current (To.5) - was voltage dependent, the current activating m o r e rapidly at depolarized potentials. T0.5 was 68.91 __ 3.78 ms (mean _+ SEM) at - 20 mV and 55.44 _+ 1.25 ms at + 20 mV (P < 0.02; paired t-test, seven cells). Activation followed third order kinetics as shown in Fig. 3A. In this cell the time constant for activation was 19 ms at 0 m V and changed e-fold every 77 mV (Fig. 3 B). Inactivation of the current could best be described as a single exponential process (Fig. 3C) with a mean time constant of 1400 __ 109 ms (n = 7) at + 20 mV. Inactivation was not voltage dependent, the time constant of inactivation being 1470 • 180 ms (n = 7) at - 2 0 inV. The time dependence of recovery from inactivation was studied as shown in Fig. 4. The cell was held at - 1 0 mV and pre-pulsed to - 9 0 m V for time periods ranging between 200 ms and 3 s before being stepped back to - 10 mV. As the duration of the pre-pulse increased so too did the amplitude of the current evoked by the test pulse (Fig. 4A). In Fig. 4B the amplitude of the peak current, normalized with respect to the current evoked following a 3.1-s pre-pulse, is plotted against prepulse duration. The fitted line is a single exponential function with a time constant of 770 ms. The current appeared similar to slowly inactivating potassium currents described in other cell types (see Discussion). In an effort to confirm this, the effect of varying the extracellular potassium ion concentration on the reversal potential of the current was studied. The reversal potential was determined from the direction of current flow during de-activating tail currents as shown in Fig. 5. The upper part of Fig. 5 A shows tail currents recorded at a range of m e m b r a n e potentials between - 7 7 m V and - 3 7 mV in the presence of 6, 10 and 20 m M extra-

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Fig. 4A, B. The time dependence of recovery from inactivation. A Currents recorded from a cell which was being held at - 10 mV and then pre-pulsed for varying durations to - 9 0 mV before being stepped back to - 10 inV. The currents shown were those activated on returning the membrane potential to - 1 0 mV. B The peak current, normalized with respect to that evoked following a 3.l-s pre-pulse, is plotted against pre-pulse duration. The fitted line is a single exponential function of the form: y = Ao + A 1[1 --exp (--t~ ~)] with a time constant (z) of 770 ms

cellular potassium. Whilst the currents shown were recorded using a voltage protocol with 10-mV voltage increments, a more accurate measurement of the reversal potential was obtained using a similar protocol but with 2-mV voltage increments covering the 10-mV potential range in which the tails reversed. The reversal potential of the current became more positive as the extracellular potassium concentration was increased. This relationship is illustrated graphically in Fig. 5 B which shows the reversal potential plotted against the logarithm of the extracellular potassium concentration. The line of best fit (r = 0.995) has a slope of 42 m V per 10-fold change in the potassium ion concentration. The dashed line is the theoretical line for a pure potassium current at 20 ~C, and has a slope of 57 m V per 10-fold change in the extracellular potassium ion concentration. Thus it appears that the current is largely, but not solely carried by potassium ions. To characterise the current further we studied its susceptibility to block by a range of drugs known to inhibit

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