Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont 05405
Submitted 14 November 2003 ; accepted in final form 14 January 2004
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ABSTRACT |
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urinary bladder excitability; membrane potential; electrophysiology; cation channels; voltage-dependent ion channel block
UBSM demonstrates spontaneous contractile activity, ascribed to depolarizing action potentials that are shaped by the presence of small- and large-conductance Ca2+-activated K+ channels, voltage-gated K+ channels, and voltage-dependent Ca2+ channels (5, 10, 11, 1318, 36). The resting membrane potential of UBSM is approximately 40 mV (8, 11, 1315, 29), which is positive to the equilibrium potential for K+ (EK; approximately 85 mV) and is critical to action potential generation (13, 14). Hyperpolarization of the membrane potential, mediated by the activation of ATP-sensitive K+ channels, reduces the frequency of UBSM action potentials and associated spontaneous UBSM contractions (6, 11, 13, 14, 25, 29, 33), indicating the requirement of a membrane potential substantially positive to EK for action potential generation in UBSM.
Despite the functional importance of the resting membrane potential in UBSM, an understanding of the ionic currents involved in generating this membrane potential is lacking. We hypothesized that a tonic, depolarizing conductance exists in UBSM, functioning to keep the resting membrane potential depolarized to a level sufficient to facilitate action potential generation. Here we identify and characterize the properties of a tonically active, Na+-permeable current in mouse UBSM (Icat), which is blocked in a voltage-dependent manner by external Ca2+, Mg2+, and Gd3+. Icat contributes tonically to the UBSM membrane potential, facilitating UBSM contractile activity.
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METHODS |
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Whole cell patch clamp. The conventional whole cell patch-clamp method was used for recordings at 22°C without the use of leak subtraction. Data were acquired with pCLAMP version 8.2 software through a 1200 series Digidata board with an Axopatch 200A amplifier (Axon Instruments, Union City, CA) filtering at 1 kHz. The physiological K+-containing bath solution was composed of (in mM) 120 NaCl, 3 NaHCO3, 4.2 KCl, 0.5 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 (NaOH), and the pipette solution contained (in mM) 110 aspartic acid (K+ salt), 30 KCl, 0.5 MgCl2, 0.77 GTP, 5 Na2ATP, 10 EGTA, and 10 HEPES, pH 7.2 (KOH) (1). In Cs+-containing solutions Cs+ was substituted for K+ in the above solutions to eliminate macroscopic K+ currents. Voltage-dependent Ca2+ channels were eliminated by 1) blocking the channels with 1 µM nifedipine and 2) inactivating the channels at a holding potential of 0 mV. The calculated equilibrium potentials for Na+, Ca2+, Cs+ (or K+), and Cl in these solutions were +64, greater than +100, 90, and 36 mV, respectively.
To differentiate effectively between the presence of cation and Cl currents in the UBSM cells, Cs+-free solutions with Na+ as the only monovalent cation, and with unique reversal potentials for Na+ (+69 mV) and Cl (82 mV), were used (34). These solutions, each 300 mosM, contained the following (in mM): bath: 120 NaCl, 50 mannitol, 10 glucose, 1.8 CaCl2, and 10 HEPES, pH 7.2 (NaOH); pipette: 120 N-methyl-D-glucamine (NMDG+), 120 aspartic acid, 50 mannitol, 5 NaCl, 1 Na2ATP, 5 EGTA, and 5 HEPES, pH 7.2 (NaOH). In ion substitution experiments NMDG+ (or Ca2+) and aspartic acid were substituted for Na+ and Cl, respectively, in the bath solution. In all experiments involving a substitution of Cl, a 1.5% agar bridge prepared in control bath solution was used.
The perforated-patch configuration of the whole cell patch-clamp technique (19, 36) was used to measure UBSM cell membrane potential and to evaluate the steady-state Icat at the resting UBSM membrane potential under physiological ionic conditions. Chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Force measurements. Tension experiments were performed as previously described (18) on mouse UBSM strips by using a MyoMed myograph system (MED Associates, Georgia, VT) with the urothelium removed. To inhibit the Na+ conductance through UBSM Icat channels Na+ in the bathing physiological saline solution was replaced with an equimolar amount of NMDG+ while other ions and pH were kept constant.
Analysis of Icat voltage-dependent block. To analyze the steady-state voltage dependence of Ca2+, Mg2+, and Gd3+ block, 1-s voltage steps were performed. Icat was measured as the difference between the current at a given concentration of blocking ion and the current measured in the same cell during bath replacement of Na+ with NMDG+, thereby providing for a pure measure of Icat without contaminating leak current. For experiments with Mg2+ and Gd3+, Icat values were normalized to control (presence of 10 µM external Ca2+) and plotted against blocking ion concentration to obtain Ki values. For experiments with Ca2+, the magnitude of current in zero Ca2+ was extrapolated from Hill plots of averaged raw data. The zero-Ca2+ values were then used to normalize the data, and Ki values were determined from the normalized plots.
Measurements and statistical analysis.
All current measurements from individual UBSM cells were performed from average currents obtained from three to five ramp or step protocols under each experimental condition. Representative current recordings are nonaveraged, except for Figs. 68 in which averaged recordings are shown. Steady-state currents and membrane potentials were averaged over a period of 1 min, and force measurements were obtained by averaging events over a 5-min period. Data are reported as means ± SE with statistical comparisons performed via paired Student's t-tests. A P < 0.05 was deemed statistically significant.
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RESULTS |
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To investigate the ion selectivity of this conductance, Na+ (120 mM) in the bath solution was substituted with 120 mM NMDG+, a large and relatively impermeant cation, causing a negative shift in the reversal potential of the ramp current (control: 12.8 ± 1.0 mV, NMDG+: 31.4 ± 1.3 mV; P < 0.05, n = 8; Fig. 1D). Na+ substitution also reduced the inward current at 40 mV from 0.41 ± 0.06 to 0.052 ± 0.008 pA/pF (P < 0.05, n = 8; Fig. 1, D and E), indicating that a tonic depolarizing cation current is functional at the resting membrane potential of UBSM. Replacement of bath Cl with aspartic acid had no significant effect on the ramp reversal potential (control: 15.6 ± 2.2 mV, aspartic acid: 19.4 ± 1.1 mV; P > 0.05, n = 4), suggesting a dominant role of cation channels in mediating the ramp current.
Separation of cation and Cl currents in UBSM. To more effectively determine whether the ramp current recorded in Cs+-containing solutions was due to the activity of cation and/or Cl channels, Cs+-free solutions with Na+ as the only monovalent cation were used. Cs+-free solutions also demonstrated unique equilibrium potentials for Na+ and Cl (ENa = +69 mV, ECl = 82 mV; Ref. 34). Under these conditions the majority of outward ramp current between 100 and +100 mV should be due to influx of Cl and the majority of the inward current due to influx of Na+. As expected, replacement of bath Na+ with NMDG+ reduced the inward current from 0.93 ± 0.13 to 0.17 ± 0.03 pA/pF at 80 mV and shifted the reversal potential from +9 ± 4 to 34 ± 4 mV (n = 19, P < 0.05; Fig. 2). In these experiments, the Na+-sensitive difference current reversed at + 56.9 ± 5.9 mV, close to the calculated ENa (+69 mV). The inward ramp current was also reduced by 50 µM extracellular Gd3+ (control 1.5 ± 0.6 pA/pF, 50 µM Gd3+ 0.36 ± 0.07 pA/pF at 80 mV; n = 6, P < 0.05; Fig. 2), whereas the outward current at +80 mV was unaffected. These results are consistent with the presence of an inward cation current (Icat) in UBSM, mediated predominantly by Na+ influx.
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Icat can permeate Ca2+. To determine whether Ca2+ in addition to Na+ can permeate UBSM Icat channels, a comparison of ramp currents in the presence of NMDG+ and a bath solution with Ca2+ as the sole external cation was performed. In the presence of 110 mM Ca2+, the average ramp current at 80 mV was 0.34 ± 0.06 pA/pF, significantly larger than 0.16 ± 0.03 pA/pF in the presence of NMDG+ (P < 0.05, n = 6; Fig. 3) and indicating an ability of UBSM Icat to permeate Ca2+ in the absence of Na+.
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Block of Icat by Mg2+, Ca2+, and Gd3+ is voltage dependent.
A comparison of UBSM Icat, measured as the Na+-sensitive current (Fig. 4B), to the Ca2+- or Gd3+-sensitive currents (Fig. 4, A and C, respectively) suggested that polyvalent cation block of Icat was greater at more negative voltages. Therefore, to measure steady-state block of Icat by Mg2+, Ca2+, and Gd3+ at different voltages, we used a voltage step protocol elicited from a holding potential of 0 mV, stepping to voltages between 100 and 20 mV and varying the concentration of blocking ion in the bath solution, which contained 10 µM Ca2+. Figure 6A shows a representative recording of a family of currents elicited by this protocol before and after 50 µM Mg2+. End-pulse currents at each voltage were then normalized to control values (I/Icontrol) and plotted against the concentration of Mg2+ (Fig. 6B). The data were then fit with a Hill equation
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Analogous experiments were performed with Ca2+, yielding Ki values of 17.6, 19.9, 37.6, 115, and 255 µM at 100, 80, 60, 40, and 20 mV, respectively (Fig. 7, A and B). Plotting Ki vs. membrane potential yielded Ki(0) of 0.94 mM, Ki-min of 15 µM, and µ of 0.81 for Ca2+ (Fig. 7C).
Experiments with Gd3+ demonstrated Ki values of 0.40, 0.48, 0.68, 1.3, and 3.4 µM at 100, 80, 60, 40, and 20 mV, respectively. Plotting Ki values vs. membrane potential yielded Ki(0) of 9.4 µM Gd3+, Ki-min of 0.38 µM, and µ of 0.49 under these conditions (Fig. 8).
In these experiments a time-dependent decline in inward Icat during hyperpolarizing voltage steps was not observed with concentrations of Mg2+ and Ca2+ in the bathing solution that provided detectable block (Figs. 6 and 7). However, a time-dependent block of UBSM Icat by Gd3+ was observable, as predicted by the low micromolar Ki values (Fig. 8). As expected for a bimolecular interaction of Gd3+ with the channel, the time course of the onset of current block was best fit with a single exponential function during voltage steps from a holding potential of 0 mV to 100, 80, and 60 mV (Fig. 8D). These voltages were chosen to yield a significant amount of inward Icat, so as to facilitate the measurement of the onset of Gd3+ block. In the same manner, 0.5 and 2.5 µM concentrations of Gd3+ were selected to yield a significant degree of block of UBSM Icat (Fig. 8). As expected, the time constants () for Gd3+ block were significantly faster for 2.5 µM compared with 0.5 µM Gd3+ (Fig. 8D), indicating a faster onset of block with a higher concentration of Gd3+. There was no significant difference between the
values at 100, 80, or 60 mV at each concentration of Gd3+ (Fig. 8D).
The of the onset of block is related to the concentration of Gd3+ by the following linear equation
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The Ki for Gd3+ block can alternatively be estimated by the equation
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Role of Icat in maintaining UBSM membrane potential. To evaluate the physiological role of Icat in the regulation of UBSM membrane potential we used the perforated-patch configuration of the whole cell patch-clamp technique, with physiological ionic gradients that included the presence of K+. Voltage-clamp experiments were performed to measure the steady-state Icat at the resting UBSM membrane potential (40 mV), and current-clamp experiments were performed to evaluate the contribution of Icat to the UBSM cell membrane potential (Fig. 9).
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Under current-clamp conditions the average membrane potential of UBSM cells was 36.5 ± 3.7 mV (n = 5). This value is consistent with the resting membrane potential reported in previous studies in which microelectrodes were used to record UBSM membrane potential (11, 1315, 29). Under current clamp, replacement of bath Na+ with NMDG+ caused a 19.6 ± 2.6-mV hyperpolarization, resulting in an average membrane potential in the presence of NMDG+ of 56.0 ± 2.3 mV (n = 5; Fig. 9, C and D). Analogous to the effects of NMDG+ on the holding current under voltage clamp, the membrane potential measured under current clamp was rapidly restored to control levels by a return to the Na+-containing bath solution (Fig. 9, C and D). Together these data suggest a dominant role of Na+-permeable Icat in the maintenance of the UBSM membrane potential.
Modulation of UBSM contractility by Icat. To further elucidate the functional role of Icat in UBSM we performed contractility experiments replacing bath Na+ with NMDG+ to inhibit Icat. Inhibition of tonically active Icat should hyperpolarize the membrane potential of UBSM strips and thereby decrease contractility. Because of the low phasic contractility demonstrated by UBSM strips isolated from the mouse we induced tone in the strips with the muscarinic agonist carbachol, which increases the level of baseline force as well as the amplitude and frequency of UBSM phasic contractions (Fig. 10; Ref. 28). In the continued presence of carbachol, reduction of bath Na+ with NMDG+ resulted in a decrease in UBSM contractility. Baseline force as well as the amplitude and area of phasic contractions were reduced (n = 7; Fig. 10).
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DISCUSSION |
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Voltage-dependent ionic block of UBSM Icat. We characterized the blocking of UBSM Icat channels by Ca2+, Mg2+, and Gd3+. The block of Icat by external divalent and trivalent cations is voltage dependent, decreasing steeply positive to 40 mV and saturating negative to 60 mV.
Voltage-dependent block of UBSM Icat by Ca2+, Mg2+, and Gd3+ bears similarity to the block of arterial smooth muscle inward rectifier K+ channels (Kir) by Cs+ and Ba2+ (30, 31). In contrast to ionic block of UBSM Icat, block of arterial Kir by Cs+ and Ba2+ does not demonstrate a saturation of the voltage dependence at negative voltages.
The on rate for Ca2+ and Mg2+ block of UBSM Icat is likely to be rapid (<10 ms) at all concentrations of blocking ion that provide a measurable amount of block, because a time-dependent onset of block by Ca2+ and Mg2+ was not observed. This is similar to Cs+ block of arterial Kir (31). However, this contrasts with the observable time-dependent Gd3+ block of UBSM Icat (Fig. 8) and Ba2+ block of smooth muscle Kir channels (30, 31). The data presented here for UBSM Icat suggest similarity in the blocking mechanism for all three polyvalent cations examined. The saturation observed in the voltage-dependent blocking mechanism of UBSM Icat at negative voltages could reflect permeation of the blocking cations through Icat channels at high driving forces.
The rapid block of UBSM Icat by Ca2+ and Mg2+ presumably reflects the rapid on rate due to high concentrations of the blocking ions. Interestingly, this is consistent with the fast external Mg2+ block demonstrated for heterologously expressed TRPV6 channels [a member of the transient receptor potential (TRP) superfamily of cation channels; Ref. 9] that are also blocked by external Mg2+ in a similar concentration range (compare Ref. 37 and Fig. 6). The Ki for external Mg2+ block of TRPV6 also decreases steeply at negative voltages and saturates with large driving forces, reaching a minimum Ki value for Mg2+ block in the micromolar range (37), analogous to UBSM Icat (Fig. 6). External Mg2+ block of TRPV6 was relieved at very high driving forces and was interpreted as a "punch through" of Mg2+ permeating the channels (37). This is consistent with our interpretation of the data reported here for UBSM Icat. Icat demonstrates an ability to permeate Ca2+ (Fig. 3), and in addition Ca2+ and Mg2+ block of UBSM Icat yields µ values close to 1 (Figs. 6 and 7), indicating that divalent ions may sense the entire electric field across the membrane and permeate the length of the UBSM Icat pore during block. Alternatively, a steep voltage dependence positive to 40 mV might reflect the properties of a multisite pore. Block of UBSM Icat channels by polyvalent cations demonstrates considerable similarity to TRPV6 of the TRP superfamily.
Block of the TRP superfamily member TRPM7 [suggested to underlie the Mg2+-inhibited current (MIC) in a number of cell types; see, e.g., Ref. 23] by external Mg2+ and polyamines also demonstrates a blocking mechanism analogous to that of UBSM Icat and TRPV6, with a saturating voltage dependence and relief of block with very large driving forces (21). The voltage-dependent blocking mechanism described here for polyvalent cations may be common to numerous members of the TRP superfamily and may be a function of an ability of these channels to permeate various polyvalent cations. It therefore seems likely that members of the TRP superfamily encode the genes responsible for the molecular basis of UBSM Icat. Consistent with this, we have identified numerous mRNA transcripts for TRP superfamily members expressed in mouse UBSM (Phillips JP, Thorneloe KS, Morita H, and Nelson MT, unpublished observations).
Smooth muscle nonselective cation channels. A number of nonselective cation currents have been identified in smooth muscle, including 1) receptor-operated channels activated by excitatory agonists signaling through heptahelical receptors or via direct channel gating (24), 2) store-operated channels activated by depletion of intracellular Ca2+ stores (2), and 3) stretch- or swelling-activated channels that respond to increases in pressure (38, 39). Although these cation channels contribute to the effects of various excitatory stimuli in smooth muscle, cation channels not requiring stimulation for activity have only recently been identified.
In rat UBSM cells an inward cation current demonstrating a slow time-dependent activation (time constant 200 ms at 100 mV) on hyperpolarization and block by 1 mM Cs+ has been reported (12). Green and colleagues (12) concluded that this cation current was similar to the hyperpolarization-activated cation current (Ih) extensively characterized in cardiac myocytes and neurons (32). Mouse UBSM Icat, identified here, is significantly different from Ih in rat UBSM, because it activates in a time-independent manner with membrane hyperpolarization (Figs. 68) and demonstrates an insensitivity to blockade by 2 mM external Cs+ (n = 3). Moreover, Green and colleagues (12) used leak subtraction in their study, eliminating any time-independent background currents, such as the UBSM Icat described here.
Few reports identifying the presence of tonically active, time-independent cation currents in freshly isolated smooth muscle cells have been made. These have been limited to vascular smooth muscles of the rabbit, including ear artery (3), pulmonary artery (4), and aorta (40, 41). In these studies the tonic cation currents demonstrated an inhibition by extracellular Ca2+ and Mg2+. Here we have extended this observation to mouse UBSM, a visceral smooth muscle. In addition, we have characterized the blocking mechanism underlying the inhibitory effect of Ca2+ and Mg2+, demonstrating that the block decreases steeply over the range of the action potential (40 to 0 mV) and becomes voltage independent at membrane potentials negative to 60 mV. We demonstrate that the trivalent cation Gd3+, used as a nonselective cation channel inhibitor, shares a similar blocking mechanism with Ca2+ and Mg2+. We were also able to estimate the on and off rates for Gd3+ at 107 M1·s1 and 310 s1, respectively. In intact smooth muscle preparations removal of external Ca2+ has been demonstrated to depolarize taenia coli (7) and guinea pig UBSM (26). It seems likely that currents similar to the mouse UBSM Icat described here exist in a number of smooth muscles, both visceral and vascular, providing a tonic depolarizing influence that regulates the resting membrane potential.
Physiological role of Icat channels in UBSM. The maintenance of the resting membrane potential in UBSM (40 mV) is critical to the generation of action potentials, which underlie spontaneous contractile events in the urinary bladder. This has been demonstrated by the activation of ATP-sensitive K+ channels evoking hyperpolarization of the resting membrane potential and a reduction in action potential and spontaneous contraction frequency (6, 11, 13, 14, 25, 29, 33). Voltage-dependent Ca2+ channels are required to mediate the upstroke of the UBSM action potential, but blockade of voltage-dependent Ca2+ channels has little effect on the resting membrane potential (1315). Our data suggest that the tonic depolarizing activity of Icat contributes to the maintenance of UBSM membrane potential (Fig. 9) and functions to enhance UBSM contractility (Fig. 10). In this capacity UBSM Icat contributes to the maintenance of urinary bladder tone.
In addition, during the depolarizing phase of the UBSM action potential block of UBSM Icat by external Ca2+ and Mg2+ will be relieved, effectively increasing the depolarizing Na+ current through Icat channels (Figs. 6 and 7). The Ki for Ca2+ and Mg2+ block of Icat increases by 8- and 28-fold, respectively, during depolarization from the UBSM resting membrane potential of 40 mV to the peak of the action potential at 0 mV. This dramatic change in Ki, combined with the fast off rate for Ca2+ and Mg2+ as low-affinity blockers (estimated to be on the order of microseconds based on the measured Ki values), would result in a substantial reduction in Icat block and an increase in the open probability of Icat channels, possibly facilitating depolarization of the action potential. The precise contribution of Icat to the action potential would depend on the relative K+/Na+ selectivity.
In summary, we have identified a tonically active, Na+-permeable current, Icat, in mouse UBSM cells that is blocked in a voltage-dependent manner by externally applied Ca2+, Mg2+, and Gd3+. Our data provide evidence that under physiological conditions the tonic, depolarizing influence of Icat channels plays an important role in the maintenance of the resting membrane potential functioning to increase the contractility of UBSM.
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GRANTS |
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ACKNOWLEDGMENTS |
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Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, College of Medicine, Univ. of Vermont, Burlington, VT 05405 (E-mail: Mark.Nelson{at}uvm.edu).
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