Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

Robert H. Cox1 and Steven Petrou2

1 Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085; and 2 Department of Physiology, University of Melbourne, Parkville, Australia 3052


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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These experiments were performed to determine the effects of reducing Ca2+ influx (Cain) on K+ currents (IK) in myocytes from rat small mesenteric arteries by 1) adding external Cd2+ or 2) lowering external Ca2+ to 0.2 mM. When measured from a holding potential (HP) of -20 mV (IK20), decreasing Cain decreased IK at voltages where it was active (>0 mV). When measured from a HP of -60 mV (IK60), decreasing Cain increased IK at voltages between -30 and +20 mV but decreased IK at voltages above +40 mV. Difference currents (Delta IK) were determined by digital subtraction of currents recorded under control conditions from those obtained when Cain was decreased. At test voltages up to 0 mV, Delta IK60 exhibited kinetics similar to control IK60, with rapid activation to a peak followed by slow inactivation. At 0 mV, peak Delta IK60 averaged 75 ± 13 pA (n = 8) with Cd2+ and 120 ± 20 pA (n = 9) with low Ca2+ concentration. At test voltages from 0 to +60 mV, Delta IK60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20 mV. At +60 mV, the initial peak Delta IK60 averaged 115 ± 18 pA with Cd2+ and 187 ± 34 pA with low Ca2+. With 10 mM pipette BAPTA, Cd2+ produced a small inhibition of IK20 but still increased IK60 between -30 and +10 mV. In Ca2+-free external solution, Cd2+ only decreased both IK20 and IK60. In the presence of iberiotoxin (100 nM) to inhibit Ca2+-activated K+ channels (KCa), Cd2+ increased IK60 at all voltages positive to -30 mV while BAY K 8644 (1 µM) decreased IK60. These results suggest that Cain, through L-type Ca2+ channels and perhaps other pathways, increases KCa (i.e., IK20) and decreases voltage-dependent K+ currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

L-type calcium channels; vascular smooth muscle; mesenteric arteries; electrophysiology; patch clamp


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AGONIST ACTIVATION of smooth muscle initiates a complex sequence of excitation-contraction coupling events (36). These include coupling of cell surface receptor occupancy to the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate by a specific phospholipase C, with the resultant production of inositol trisphosphate (IP3) (2), the release of intracellular Ca2+ stores (26), myosin light chain phosphorylation (20), and rapid force development (11). Additional mechanisms come into play to sustain contraction, despite the fact that the increases in IP3, cytosolic free Ca2+, and myosin light chain phosphorylation are largely transient (2, 11, 26). The mechanisms responsible for force maintenance are less well understood than those responsible for force development but have been suggested to involve protein kinase C activation (19, 21) and/or an increase in Ca2+ sensitivity of the contractile proteins (5, 32).

Force maintenance has been shown to have a critical dependence on extracellular Ca2+ influx as well as a close coupling to membrane potential (17, 27, 37). In the absence of extracellular Ca2+, contractile responses to agonists cannot be maintained (9, 34). Agonist activation causes membrane depolarization, with subsequent activation of voltage-dependent Ca2+ influx (29), and also indirectly activates nonselective cation channels (33, 41).

Mechanisms contributing to the depolarization of the membrane potential by agonists have only recently been identified. Ca2+ released from the sarcoplasmic reticulum (SR) by agonists activates Ca2+-sensitive Cl- channels (ClCa) (41) and inhibits voltage-dependent K+ channels (Kv) (13). Both of these effects are thought to contribute to the membrane depolarization. There is direct evidence that membrane depolarization is sustained for the duration of agonist exposure on the basis of membrane potential measurements (27). Furthermore, there is indirect evidence based on the observation that organic Ca2+ channel blockers can inhibit sustained agonist-induced contractions in arterial smooth muscle (31), especially in resistance arteries (4) and in arteries from hypertensive subjects (1).

Considering the fact that SR Ca2+ release is transient (26) and ClCa currents inactivate with sustained increases in cytosolic Ca2+ concentration ([Ca2+]) (43), the question arises as to how the membrane depolarization associated with agonist activation is sustained. Recently, it was demonstrated that L-type Ca2+ channels (CaL) exhibit measurable open probability under steady-state conditions at voltages between -40 and -20 mV in arterial smooth muscle (35). It has also been shown that a voltage "window" exists for CaL activation, which produces a similar voltage window of sustained, elevated cytoplasmic [Ca2+] (12). These results suggest that sustained Ca2+ influx through CaL associated with membrane depolarization may also provide a mechanism to inhibit Kv. It was the purpose of the experiments reported in this study to test the hypothesis that Ca2+ influx can inhibit Kv and provide a mechanism to produce sustained membrane depolarization during agonist activation, thereby contributing to force maintenance.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Cells were isolated from rat small mesenteric artery branches using techniques previously described in detail (6, 8). Briefly, individual branches were cut open longitudinally and incubated at 37°C for 60 min in a Ca2+-free buffer. The segment was then cut into small pieces and incubated in buffer with collagenase (250 U/ml) and elastase (25 U/ml) for ~30 min without agitation at 37°C. The tissue was gently aspirated with a Pasteur pipette, and released cells were separated by filtration through 210-µm nylon mesh.

Electrophysiology. An aliquot of cells was placed in a perfusion chamber mounted on an inverted microscope (Nikon Diaphot), allowed to adhere to the glass bottom, then superfused with a HEPES-buffered solution. The external [Ca2+] was slowly increased to 2 mM to avoid damaging the cells. Cells were sealed to pipettes with gentle suction, and the membranes were ruptured by negative pressure at a holding potential of -40 mV. Membrane currents were recorded using whole cell or perforated patch-clamp techniques (18) with a voltage-clamp amplifier (model 8900; Dagan). Fire-polished micropipettes (2-3 MOmega resistance) were fashioned from capillary tubing (Kwik-fil; WPI) using a micropipette puller (model P-80/PC; Sutter Instruments). Series resistance and stray capacitance compensation were employed using the amplifier circuitry. For perforated-patch conditions, junction potential was compensated before measurements. Current and voltage signals were converted from analog to digital form at a sampling rate of 2 kHz (Labmaster A/D board) and stored in a computer (Gateway 2000 486DX) for subsequent analysis.

Protocols. Whole cell currents were measured using both voltage-ramp and voltage-step protocols from holding potentials of -60 and -20 mV. With the ramp protocol, voltage was increased at 1 mV/ms from either holding potential to a maximum value of +60 mV. With the step protocol, 1-s voltage steps were applied from -60 to +60 mV from both values of holding potential in 10-mV increments at intervals of 15 s. For statistical analysis and comparisons, peak currents and currents averaged over the last 100 or 200 ms of each voltage-clamp step were determined. For most conditions, current responses were recorded before and during an intervention. Difference currents were determined between such conditions by subtracting digital current records obtained under control conditions from those obtained during an intervention at each value of test voltage. Current records were analyzed using pCLAMP software (version 5.5.1; Axon Instruments).

Chemicals and solutions. Collagenase was purchased from Worthington Biochemical, Freehold, NJ (type CLS3), and elastase was from ICN Nutritional Biochemicals, Cleveland, OH (hog pancreas). All other chemicals were obtained from Sigma Chemical, St. Louis, MO. Water (>18 MOmega ) was obtained from a Barnstead purification system with an organic final filter. The solution used for myocyte isolation contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 10 dextrose at pH 7.4 (with NaOH). The external perfusion solution was the same, with 2 mM Ca2+ added. The pipette solution for whole cell studies contained (in mM) 140 KCl, 5 NaCl, 5 MgATP, 10 HEPES, and 0.2 or 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at pH 7.2 (with KOH) and had a resistance of 2.5-3.5 MOmega . HEPES was increased with the 0.2 mM BAPTA internal solution to maintain osmolarity constant at ~300 mosM. The pipette solution for perforated-patch studies contained (in mM) 110 potassium gluconate, 30 KCl, 5 NaCl, 0.1 CaCl2, and 10 HEPES with 150 µg/ml amphotericin at pH 7.2 (with KOH).

Statistical analysis. Statistical comparisons of membrane currents were performed using a two-way ANOVA with repeated measures for paired data using the STATWORKS application on a Power Macintosh computer. Probability values of <0.05 were considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the first series of experiments, the effects of 0.1 mM Cd2+ (to inhibit CaL) on K+ current (IK) were determined. Figure 1 shows current responses to voltage ramps from each holding potential before and during the addition of 0.1 mM Cd2+ to the perfusate. When IK was recorded from a holding potential of -60 mV (IK60), Cd2+ increased net outward current at voltages just above the activation threshold (Fig. 1A). At more positive voltages up to +60 mV, however, net outward current was decreased with Cd2+. When recorded from a holding potential of -20 mV (IK20), Cd2+ only decreased net outward current at voltages >0 mV (Fig. 1B). Difference currents, obtained by digital subtraction of current ramps before and during Cd2+, also shown in Fig. 1, confirm this description. We initially assumed that, under the conditions of these experiments, K+ currents dominate these responses. Whole cell K+ currents have been shown in many smooth muscle cells to be composed primarily of Kv and Ca2+-activated K+ channel (KCa) components (22, 29). Because Kv currents are expected to be inactivated (decreased availability) at a holding potential of -20 mV (22, 46), the IK20 data should primarily represent the KCa component. The above IK20 results suggest that inhibiting CaL with Cd2+ reduced KCa current. Because both Kv and KCa currents are expected to contribute to IK60, and since Cd2+ inhibits IK20 (i.e., KCa) only at voltages >0 mV, one interpretation of the IK60 responses at voltages <0 mV is that Kv currents are augmented in the presence of Cd2+. The presence of both Kv and KCa components of IK60 were observed in every cell studied.


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Fig. 1.   Effects of external Cd2+ on current responses to voltage ramps. Current responses were recorded from holding potentials of -60 mV (A) and -20 mV (B). Arrows, beginning of voltage ramp. Each panel shows control ramp responses before () and during addition of 0.1 mM Cd2+ to external perfusion solution. Below these responses are difference currents obtained by digital subtraction of ramps obtained with Cd2+ minus those before Cd2+. Holding current before and after ramps are included as baseline references. Calibration bars in A apply to all currents and represent 200 pA and 100 ms, respectively.

Current responses to voltage ramps are only qualitative at best, since voltage-dependent activation and inactivation kinetics may obscure the details of currents recorded by this method. To study the effects of Cd2+ on IK quantitatively, measurements of IK were made in response to voltage steps from holding potentials of -60 and -20 mV with 2 mM external [Ca2+] and 0.2 mM pipette BAPTA in the absence then the presence of 0.1 mM Cd2+. Figure 2 shows representative results of whole cell currents recorded at two values of test potential (0 and +60 mV) from the two values of holding potential (-60 and -20 mV). The addition of Cd2+ to the perfusion solution consistently increased IK60 at a test voltage of 0 mV (Fig. 2A, top) but decreased IK60 at a test voltage of -60 mV (Fig. 2C, top) compared with control conditions. Inspection of the current responses suggests that the kinetics of the current was also altered by Cd2+. Therefore, difference currents [Delta IK60(Cd)] were calculated between the two conditions. At 0 mV test voltage, Delta IK60(Cd) increased rapidly to a peak and subsequently declined to a steady level that was maintained for the duration of the voltage step (Fig. 2A, bottom). The peak value of Delta IK60(Cd) averaged 75 ± 13 pA, whereas the "steady" level (last 100 ms of the clamp step) averaged 31 ± 9 pA (n = 8). Although the majority of Delta IK60(Cd) recorded at the +60-mV test voltage was negative (smaller net outward current), there was an early positive phase for the difference current (arrow in Fig. 2C, bottom). The peak value of this early current averaged 115 ± 18 pA, whereas the steady level averaged -148 ± 52 pA (n = 8) at +60 mV. When recorded at a test voltage of 0 mV from a holding potential of -20 mV, the currents were very small (Fig. 2B, top) and the difference current [Delta IK20(Cd)] was essentially zero. When measured at a test potential of +60 mV (Fig. 2D, bottom), Delta IK20(Cd) was consistently decreased in the presence of Cd2+ and never exhibited an early positive peak as found in Delta IK60(Cd). The steady level of Delta IK20(Cd) averaged over the last 200 ms of the voltage-clamp step was -288 ± 48 pA (n = 8).


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Fig. 2.   Effects of Cd2+ on K+ current (IK) measured with 2 mM external [Ca2+] and 0.2 mM pipette BAPTA. Currents were measured from holding potentials of -60 (A and C) or -20 mV (B and D). In each panel, currents before () and during addition of 0.1 mM Cd2+ to the perfusion solution are shown at test voltages of either 0 mV (A and B) or +60 mV (C and D). Difference currents obtained by digital subtraction of current with and without Cd2+ are shown in each panel. Calibration bars represent 60 pA (A and B) or 200 pA (C and D) and 100 ms (A-D).

To expand on these observations, measurements were repeated at several test potentials from both values of holding potential (i.e., IK20 and IK60). Examples of difference currents determined from currents measured before and during Cd2+ at various values of test potential are shown in Fig. 3. Over a test potential range from -20 to +20 mV (Fig. 3A), Delta IK60(Cd) rose to an initial peak that increased with test voltage then appeared to "inactivate" with time. This secondary decline increased with increasing test voltage and was also associated with an increase in current "noise." With further increases in test potential >20 mV, the initial peak became shorter in duration and was followed by a net negative outward current. Delta IK60(Cd) always exhibited an initial positive phase at all test potentials, but values during the sustained phase of the voltage-clamp step exhibited a transition from positive values at negative test potentials to negative values at positive test potentials. This behavior was different from that recorded from a holding potential of -20 mV (Fig. 3B). Values of Delta IK20(Cd) were zero at all voltages up to 0 mV (Fig. 3B). At test potentials >0 mV, values of Delta IK20(Cd) were only negative (decreased net outward current or increased net inward current) over the entire duration of the voltage-clamp step. Delta IK20(Cd) never demonstrated an early positive value.


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Fig. 3.   Voltage dependence of difference currents (Delta IK) associated with addition of Cd2+ to perfusate. A: Delta IK determined from IK recorded with and without Cd2+ at test voltages from -20 to +60 mV as indicated from a holding potential of -60 mV. B: similar data recorded from a holding potential of -20 mV. Responses from a -60 mV holding potential develop a biphasic character with increasing test voltage (i.e., positive to negative values), whereas those from a -20 mV holding potential are only monophasic (negative). Calibration bars represent 400 pA and 100 ms, respectively.

Peak values and steady values of Delta IK60(Cd) averaged over the last 100 ms of the voltage-clamp step were determined at different test potentials, and average results are shown in Fig. 4A. Peak values of Delta IK60(Cd) "activated" at about -30 mV and increased with voltage up to +60 mV. Steady values of Delta IK60(Cd) also activated at about -30 mV, increased to a maximum value at about +10 mV, then decreased with increasing test potential, becoming negative at about +40 mV and higher. Steady values of Delta IK20(Cd) were determined over the last 200 ms of the voltage-clamp step, and results are summarized in Fig. 4B. Steady values of Delta IK20(Cd) were significantly different from zero only at voltages >0 mV, where they were always negative (smaller net outward current). Steady values of Delta IK20(Cd) were significantly larger (i.e., more negative) than steady values of Delta IK60(Cd) at all voltages >0 mV. These results are qualitatively similar to those presented in Fig. 1 for ramp current responses.


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Fig. 4.   Summary of voltage dependence of difference currents measured in presence of Cd2+ from holding potential of -60 mV [Delta IK60(Cd)] (A) and -20 mV [Delta IK20(Cd)] (B). A: responses determined from peak value of Delta IK60(Cd) (open circle ) and value averaged over last 100 ms of voltage-clamp step (). B: value of Delta IK20(Cd) averaged over last 200 ms of voltage-clamp step. Symbols are means and vertical lines are ±1 SE (n = 8).

The current responses at the two holding potentials have been interpreted above based on the assumption that only KCa currents contribute to IK20, whereas both Kv and KCa contribute to IK60. The validity of the conclusions concerning the effects of Cd2+ on IK components rests in part on the validity of this assumption. Therefore, experiments were performed to determine the effects of blockers of Kv (4-aminopyridine) and KCa (iberiotoxin) on IK20 and IK60 to test this assumption. As shown in Fig. 5, 100 nM iberiotoxin completely inhibited IK20, whereas it had a relatively small effect on IK60 (Fig. 5B), including reducing the current noise. In the presence of iberiotoxin, prominent tail currents remain in IK20, representing the recovery of Kv from inactivation following clamp steps to voltages negative to the -20-mV holding potential. When 1 mM 4-aminopyridine was added with iberiotoxin, IK60 was reduced substantially, as was the tail current in IK20 (Fig. 5C). Similar results were obtained in six cells. These results confirm the assumption that IK20 represents primarily KCa currents, whereas IK60 represents both Kv and KCa currents.


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Fig. 5.   Pharmacology of IK recorded from mesenteric myocytes at 2 holding potentials. Families of IK were recorded from holding potentials of -60 (top traces) and -20 mV (bottom traces) under control conditions (A), after addition of 100 nM iberiotoxin to perfusate (B), and after addition of 1 mM 4-aminopyridine with iberiotoxin (C). Calibration bars below C represent 300 pA and 400 ms.

If the responses shown in Figs. 1-4 are the result of inhibiting Ca2+ influx on IK components by Cd2+ as suggested, then it would be expected that decreasing Ca2+ influx by lowering external [Ca2+] should have similar effects. The effects of decreasing external [Ca2+] from 2 to 0.2 mM on IK were determined to test this expectation. External [Mg2+] was simultaneously increased to 2.8 mM to maintain divalent ion concentration constant. The results are summarized in Fig. 6. At a test potential of 0 mV, decreasing external Ca2+ increased IK60, and the difference current, Delta IK60(Ca), increased rapidly to a peak then declined slowly for the duration of the clamp step (Fig. 6A). The peak value of Delta IK60(Ca) at 0 mV test voltage averaged 120 ± 20 pA while the steady value averaged over the last 100 ms of the clamp step was 59 ± 19 pA (n = 9). As with Cd2+, Delta IK60(Ca) recorded at a +60 mV test voltage exhibited an early positive phase (arrow in Fig. 6C) followed by a sustained negative phase. The peak value of this early current phase averaged 187 ± 34 pA while the steady level averaged -296 ± 44 pA (n = 9). When recorded from a holding potential of -20 mV, however, IK20 was only decreased when external Ca2+ was decreased, and only negative difference currents (smaller net outward currents) were observed over the entire duration of the voltage-clamp step. At a test potential of 0 mV, the difference currents were also essentially zero (Fig. 6B), but at +60 mV (Fig. 6D) Delta IK20(Ca) averaged -455 ± 78 pA. In contrast to Delta IK60(Ca), Delta IK20(Ca) never exhibited an early positive phase at a test potential of +60 mV (Fig. 6D). When the voltage dependence of the peak and late values of difference currents were determined (Fig. 6, E and F), they exhibited characteristics similar to those found with Cd2+ (Fig. 4) but with larger magnitudes.


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Fig. 6.   Effects of decreasing external Ca2+ on IK. Currents were measured from holding potentials of -60 (A, C, and E) or -20 mV (B, D, and F). Measurements were made at 0.2 and 2 mM Ca2+ () in random order. External [Mg2+] was increased by 1.8 mM in the former to maintain total divalent ion concentration constant. Current traces show representative responses and difference currents from both holding potentials at test voltages of +0 (A and B) or +60 mV (C and D). Calibration bars represent 100 pA (A and B) or 300 pA (C and D) and 100 ms (A-D). E: average values of peak (open circle ) and late () Delta IK60. F: average values of late Delta IK20 over voltage range of -60 to +60 mV. Symbols are means and vertical lines are ±1 SE (n = 9).

The positive difference currents produced by decreasing external Ca2+ could be due (in part) to a shift in the voltage dependence of IK availability as a result of altered masking of surface charge even with the compensatory increase in external [Mg2+] (15). Figure 7 shows a comparison of the effects of external [Ca2+] on steady-state inactivation of IK. There was no significant difference in the voltage dependence of this relationship when external [Ca2+] was reduced to 0.2 mM and external [Mg2+] increased to 2.8 mM. The addition of 0.1 mM external Cd2+ also had no significant effect on IK availability (data not shown). It is unlikely, therefore, that a shift in the voltage dependence of IK contributed to the effects of decreasing external Ca2+ on whole cell current described above.


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Fig. 7.   Effects of external [Ca2+] on voltage dependence of IK availability recorded with 0.2 (open circle ) and 2 mM Ca2+ () with 3 M total divalents (Ca2+ + Mg2+) in perfusion solution. Availability was determined using a 2-step protocol consisting of a 20-s conditioning voltage step (from -100 to 0 mV) followed by a 1-s step to a test voltage of 0 mV. Peak value of current recorded at each test potential was determined as a function of conditioning voltage, normalized to the maximum response range, and averaged (n = 9).

If these current responses are the result of an effect of Ca2+ influx on global intracellular [Ca2+], then IK responses to Cd2+ should be decreased in cells dialyzed against a pipette solution containing 10 mM BAPTA to buffer intracellular [Ca2+] (45). As shown in Fig. 8, responses to the addition of 0.1 mM Cd2+ to the external perfusate with 10 mM pipette BAPTA were smaller but showed some similarities to those obtained with 0.2 mM pipette BAPTA (Fig. 4). Peak values of Delta IK60(Cd) measured with 10 mM pipette BAPTA exhibited a bell-shaped voltage dependence with a peak near 0 mV test voltage (Fig. 8A). Delta IK60(Cd) at 0 mV test voltage exhibited an early positive value (increased net outward current) that averaged 68 ± 11 pA (n = 7) and inactivated more completely toward baseline over the course of the 1-s voltage-clamp step (Fig. 8A). Values of Delta IK60(Cd) with 10 mM BAPTA measured at a +60-mV test voltage did not exhibit an early positive peak, but a significantly negative difference current developed during the sustained voltage-clamp step (Fig. 8C). The late value of Delta IK60(Cd) averaged -59 ± 23 pA at a +60-mV test voltage with 10 mM pipette BAPTA. Values of IK20 recorded with 10 mM pipette BAPTA were generally smaller than those recorded with 0.2 mM pipette BAPTA as expected and were reduced by the addition of external Cd2+. These results suggest that, when cytosolic Ca2+ is dialyzed to low (nM) levels by 10 mM pipette BAPTA, blocking Ca2+ influx with external Cd2+ still increased IK60 (smaller effect compared with 0.2 mM BAPTA), but to a smaller extent and over a smaller voltage range.


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Fig. 8.   Effects of 10 mM pipette BAPTA on IK response to external Cd2+. Measurements were made before () and during addition of 0.1 mM Cd2+ to perfusate at holding potentials of -60 (A, C, and E) and -20 mV (B, D, and F). Current traces show representative responses at test voltages of +0 (A and B) or +60 mV (C and D). Calibration bars represent 100 (A and B) or 200 pA (C and D), and 100 ms. E: average values of peak (open circle ) and late () Delta IK60. F: average values of late Delta IK20 over voltage range of -60 to +60 mV. Symbols are means and vertical lines are ±1 SE (n = 7).

The direct effects of Cd2+ on IK were determined by recording responses in the absence of external Ca2+. These results are summarized in Fig. 9. IK was again measured from holding potentials of -20 (IK20) and -60 mV (IK60) with 0 mM external Ca2+ (replaced by Mg2+) and 0.2 mM pipette BAPTA. Currents measured from a holding potential of -20 mV were significantly reduced at voltages above a test voltage of +40 mV by Cd2+ (Fig. 9F), but the effect was much smaller than noted under previous conditions. At test voltages above -20 mV, Cd2+ significantly reduced currents recorded from the -60-mV holding potential (Fig. 9E). Cd2+ never increased IK under conditions of Ca2+-free perfusion.


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Fig. 9.   Effects of external Cd2+ on IK during Ca2+-free perfusion. Measurements were made before () and during addition of 0.1 mM Cd2+ to perfusate at holding potentials of -60 (A, C, and E) and -20 mV (B, D, and F). Current traces show representative responses at test voltages of 0 (A and B) or +60 mV (C and D). Calibration bars represent 100 pA and 100 ms. E: average values of peak (open circle ) and late () Delta IK60. F: average values of late Delta IK20 over voltage range of -60 to +60 mV. Symbols are means and vertical lines are ±1 SE (n = 5).

To directly test the interpretation of the previous results, experiments were performed after blocking KCa currents with 100 nM iberiotoxin. In addition, IK was recorded using the perforated-patch configuration (amphotericin) to provide more physiological conditions. The effects of inhibiting Ca2+ influx with 0.1 mM Cd2+ under these conditions are summarized in Fig. 10. When measured in the presence of iberiotoxin, the increase in IK60 (i.e., Kv) associated with the addition of Cd2+ now occurred over the entire voltage, where IK60 was active (from -30 to +60 mV). The maximum increase in IK60 averaged 100 ± 6 pA (n = 4) at a test voltage of +60 mV.


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Fig. 10.   Effects of Cd2+ on IK recorded with perforated-patch methods in presence of 100 nM iberiotoxin. A: original IK60 records were measured at 0 (left) and +60 mV (right) before () and during (open circle ) addition of 0.1 mM Cd2+ to perfusion solution. Difference currents (black-triangle) were determined by digital subtraction of those records and are shown below each pair. Calibration bars: 100 pA and 100 ms. B: peak values of difference currents were determined at each test voltage and are shown as means ± SE (n = 4). Solid curve shows a fit of the data with a Boltzmann function: peak value = 100 ± 6 pA; voltage at half-maximum current (V1/2) = 6.5 ± 2.9 mV; and slope factor = 10.4 ± 2.4 mV.

To further test the interpretation of the previous results, experiments were performed to determine the effects of increasing Ca2+ influx on IK60. IK was recorded with the perforated-patch configuration in the presence of 100 nM iberiotoxin to block KCa, and CaL current was increased by adding 1 µM BAY K 8644 to the perfusate. As shown in Fig. 11, IK60 was decreased following the addition of BAY K 8644, and the effect was reversible. The decrease in IK60 was significant at all voltages above -30 mV, where Kv was active, and had a maximum value of -97 ± 7 pA (n = 4) at a test voltage of +60 mV.


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Fig. 11.   Effects of BAY K 8644 on IK recorded with perforated-patch methods in presence of 100 nM iberiotoxin. A: families of currents measured from -60 to +60 mV shown here were recorded (from top to bottom) 1) under control conditions, 2) after addition of iberiotoxin, 3) after addition of 1 µM BAY K 8644 in presence of iberiotoxin, and 4) after washing out BAY K 8644. Calibration bars: 100 pA and 100 ms. B: peak current values determined at each test voltage before (), during (open circle ), and after (black-triangle) addition of BAY K 8644 to perfusate. C: difference currents obtained by subtracting currents recorded after addition of BAY K 8644 from those recorded before. Solid curve shows a fit of the data with a Boltzmann function: peak value = -97 ± 7 pA; V1/2 = 12.9 ± 2.8 mV; and slope factor = 14.3 ± 1.1 mV. Data are shown as means ± SE (n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of these studies demonstrate that reducing Ca2+ influx either by 1) decreasing external Ca2+ or 2) adding external Cd2+ increased steady values of IK60 at test voltages between about -40 and +10 mV but decreased IK60 at test voltages above +30 mV and decreased IK20 at test voltages above +10 mV. We have interpreted these results to indicate that decreasing Ca2+ influx relieves inhibition ("disinhibits") of Kv and decreases the activation of KCa in this tissue.

This interpretation of our experimental results based on whole cell current measurements relies on two assumptions: 1) the contribution of Kv and KCa currents to IK20 and IK60 and 2) the relative contribution of outward (primarily K+) and inward currents (primarily Cl- and Ca2+) to the net current responses. Regarding the first assumption, IK20 but not IK60 is nearly completely inhibited by iberiotoxin, a selective inhibitor of KCa, suggesting that IK20 is primarily KCa current. IK60 is only partially inhibited by iberiotoxin but is further inhibited by 4-aminopyridine, suggesting that both Kv and KCa contribute to IK60. This latter conclusion is consistent with the known steady-state availability of Kv in vascular myocytes, which have a voltage for 50% availability between -50 and -40 mV (22, 46), as well as with the known properties of KCa (10). Thus the first assumption appears to be valid.

Regarding the second assumption, the increase in difference currents associated with adding Cd2+ or reducing external [Ca2+] could be due to an increase in outward current, a decrease in inward current, or a combination of both. We have suggested that our results primarily represent changes in outward currents. However, several candidate inward currents could contribute to these responses, including Cl-, Ca2+, and nonselective cation currents (29, 33, 41, 44). Under the conditions of our experiments, both Cl- (symmetrical [Cl-]) and nonselective cation currents would be expected to have reversal potentials near 0 mV (41, 42). The results shown in Figs. 2 and 6 demonstrate that reducing Ca2+ influx by either method increased whole cell current at a test potential of 0 mV (as well as others). It is unlikely that inhibition of either Cl- or nonselective cation currents contributes significantly to the changes in IK60 at 0 mV. Furthermore, if changes in inward currents did contribute significantly to these current responses, it would be expected that the holding current at -60 mV would be increased (i.e., more positive) following inhibition of Ca2+ influx, but it was not (Fig. 1). Furthermore, the difference currents shown in Figs. 3A and 5A remain positive over the entire 1-s voltage-clamp step. It is unlikely that CaL are directly responsible for this effect, since they are likely to be small under the conditions of these experiments and they inactivate rapidly to small steady-state values (35). No voltage-gated Na+ currents or T-type Ca2+ currents are present in this tissue (8, 24). Although a minor contribution from inward currents may occur at negative test voltages, it is likely that IK60 and IK20 primarily represent contributions from Kv and KCa.

On the basis of this discussion, we have interpreted the results of these experiments to indicate that decreasing Ca2+ influx in rat small mesenteric artery myocytes augments Kv currents and inhibits KCa currents. When recorded from a holding potential of -20 mV where the dominant current is carried by KCa, decreasing Ca2+ influx decreases IK20 over the voltage range where KCa currents are active (i.e., >0 mV). When recorded from a -60 mV holding potential where both Kv and KCa currents contribute to IK60, a mixed result is predicted. Over the voltage range where Kv current dominates (negative to +10 mV), the effect of decreasing Ca2+ influx is to increase IK60. Over the voltage range where KCa current dominates (positive to +30 mV), the effect of decreasing Ca2+ influx is to decrease IK60. The results obtained in the presence of iberiotoxin confirm this interpretation. When KCa currents are inhibited, decreasing Ca2+ influx (with Cd2+) increases IK60 over the entire voltage range from -30 to +60 mV. Furthermore, increasing Ca2+ influx (with BAY K 8644) decreases IK60, as would be predicted based on our interpretation of these results. These latter two sets of experiments provide strong evidence in support of our interpretation of the results obtained in the absence of K+ channel blockers.

In apparent conflict with this simple explanation was the observation that, at positive test potentials, an initial rapid, transient positive phase of the difference current was found in Delta IK60 (Figs. 2C and 6C) but not Delta IK20 (Fig. 2D and 6D). This initial peak of Delta IK60 was followed by a secondary decrease to negative net Delta IK60 values. This biphasic response can be explained on the basis of a faster rate of activation of Kv compared with KCa currents (30, 40). At positive test potentials, the effects of Ca2+ influx on Kv and KCa contribute algebraically to the overall response. The rapid, initial transient phase represents the "disinhibition" of the more rapidly activating Kv current, but the larger inhibition of the more slowly activating KCa current subsequently dominates the Kv contribution, and the difference current declines to its final net negative value. Consistent with this explanation, when difference currents between -20 and +60 mV are inspected, a progressive transition in Delta IK60 is seen over this voltage range (Fig. 3). Although KCa dominate steady-state responses at positive test potentials, the contribution of Kv disinhibition can be seen in the initial transient current phase (Fig. 3A). However, since Kv are not available at a holding potential of -20 mV, the inhibition of KCa by decreased Ca2+ influx completely explains the observed responses of IK20 (Fig. 3B).

To test the interpretation of these results, we developed a simple model representation. We assumed that IK could be represented by the sum of Kv and KCa components. The Kv component was represented by an activation function consisting of a single exponential function to the second power (n2) plus an inactivation function consisting of two exponential components plus a noninactivating component (40). The KCa component was represented by a similar activation function. Values for the amplitude and time constants of the various functions were determined for Kv and KCa components from IK60 data obtained in the presence of iberiotoxin and from IK20 data, respectively, using the curve-fitting functions of SigmaPlot. Figure 12 shows some of the results predicted by this model at a test voltage of +60 mV. First, we assumed that reducing Ca2+ influx increased the amplitude of Kv current by 30% and decreased the amplitude of KCa current by 50%. The net effect of these changes on IK is shown in Fig. 12F. The model predicts an early, transient positive current response followed by a more slowly developing net negative current response. If only a change in KCa current is allowed to occur, the response in Fig. 12G is obtained, and there is no early positive peak, only a net negative current. This simple model accurately represents the results shown above (Figs. 2, 3, and 6) and gives support to the explanations provided. Thus the early net positive outward current peak in the composite whole cell IK response is the result of the more rapid activation kinetics of Kv compared with KCa currents.


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Fig. 12.   Model representation of effects of Ca2+ influx inhibition on IK. A: voltage-dependent K+ channel (Kv) activation represented by function [1 - exp(-t/0.01)]2. B: Kv inactivation represented by {0.25 + 0.25[exp(-t/0.25)] + 0.5[exp(-t/2)]}. C: Kv represented as product of activation and inactivation terms (A × B). D: Ca2+-activated K+ channel (KCa) activation represented by [1 - exp(-t/0.03)]2. E: total IK represented as sum of Kv and KCa components (C + D). F: IK difference current (IK before minus IK after) decreasing Ca2+ influx, assuming Kv current is increased by 30% and KCa current is decreased by 50%. G: IK difference current, assuming only a 50% inhibition of KCa current. Time constants are in seconds and were determined from experimental data recorded at a test voltage of +60 mV. IK component amplitudes were normalized to produce peak values of one. Calculations were performed using Excel (Microsoft, Redwood, WA) and plotted using SigmaPlot (Jandel, San Rafael, CA).

How does Ca2+ influx contribute to these effects on IK components? The results obtained with 10 mM pipette BAPTA suggest that CaL current activated by voltage-clamp steps directly contributes to these responses. The primary evidence to support this conclusion comes from the voltage dependence of the peak value of Delta IK60(Cd) recorded under these conditions, which mirror the voltage dependence of CaL currents (7, 8). However, the voltage dependence of peak Delta IK60(Cd) or Delta IK60(Ca) recorded with 0.2 mM pipette BAPTA does not exhibit a similar voltage dependence. Instead of exhibiting a bell-shaped variation with voltage peaking near 0 mV, these responses extend to positive test potentials, where they achieve their largest values. This is particularly apparent in the results obtained in the presence of KCa blocked by iberiotoxin (Fig. 10). This suggests that the time-dependent CaL is not the primary determinant of the IK responses to decreased Ca2+ influx under more normal conditions (0.2 mM pipette BAPTA). It is likely, therefore, that a finite open probability of CaL contributes significantly to global intracellular Ca2+ at a holding potential of -60 mV (35). This conclusion is supported by the observation that augmentation of CaL by BAY K 8644 inhibits Kv over the entire test voltage range up to +60 mV. The greater effect of time-dependent CaL in the presence of 10 mM BAPTA is likely due to the much larger current amplitude that is expected when global intracellular Ca2+ is reduced, resulting in the loss of Ca2+-induced inactivation of CaL (15)

We chose to use Cd2+ to inhibit CaL in these experiments, because in initial studies we found that 100 nM nisoldipine inhibited IK60 with 0.2 mM external Ca2+ and 0.2 mM pipette BAPTA. Under these conditions, it is expected that only a small CaL would exist, so that the inhibition of IK by nisoldipine probably represents a direct inhibitory effect on Kv currents. Data in the literature demonstrate that heterologously expressed channels of the Shaker family are inhibited by dihydropyridines (16). A similar effect of dihydropyridines has been reported in rabbit coronary artery myocytes (22).

The effect of Cd2+ on IK60 in the voltage range from -20 to +10 mV was similar with 0.2 or 10 mM pipette BAPTA (Figs. 2, 4, and 8). With the higher pipette BAPTA it would be expected that global intracellular Ca2+ would be much lower but that the Ca2+ current would be larger because of a reduction in Ca2+-induced inactivation of CaL (3). These results could be interpreted to suggest that CaL and Kv may be colocalized in the plasma membrane in a region not readily accessible to BAPTA or in which free diffusion of Ca2+ may be restricted. The difference current recorded with 10 mM BAPTA exhibited a continuous decrease with time, consistent with slow diffusion and/or buffering of local Ca2+. Similar suggestions have been made concerning the colocalization of functional elements involved in smooth muscle excitation-contraction coupling, including SR Ca2+-release channels and plasma membrane KCa (28), plasma membrane Na+ pump and Na+/Ca2+ exchanger, and calsequestrin-containing regions of the SR (25).

The effects of Ca2+ influx on whole cell IK are similar to those demonstrated for the IK response to SR Ca2+ release mediated by either agonists or caffeine (13, 14). These investigators further demonstrated that Ca2+ added at micromolar levels to the cytoplasmic surface of inside-out patches inhibited the open probability of single-channel events by decreasing mean open time. Similar but more complicated results have been described for the effects of Ca2+ on inside-out patches of Xenopus oocytes expressing colonic smooth muscle Kv1.5 channels (39).

It has been known for some time that agonist activation of smooth muscle produces membrane depolarization (17, 27, 37). Ca2+ influx is then increased as a result of an increase in open probability of CaL associated with the membrane depolarization (29) and as a direct result of the effect of the agonist on CaL (23). Activation of ClCa (33) and inhibition of Kv (13) by SR Ca2+ release have both been suggested to participate in this response. This has been suggested to be a positive-feedback mechanism to maintain Ca2+ influx and elevated cytosolic Ca2+ (13). However, it is known that agonists, in addition to promoting SR Ca2+ release, maintain the release channels in the open configuration, preventing subsequent SR Ca2+ accumulation and release, thereby allowing Ca2+ influx to sustain contraction (38). The results of the present experiments suggest that Ca2+ influx can also inhibit Kv, thereby sustaining the membrane depolarization and the increased open probability of CaL. ClCa have been shown to inactivate during sustained increases in cytosolic Ca2+ (44) by a Ca2+/calmodulin kinase II-mediated process (43). This suggests that, while an action of SR Ca2+ release on ClCa and Kv may be involved in the initial phase of force development, sustained membrane depolarization and CaL influx may be maintained by an action of Ca2+ influx on Kv, contributing to force maintenance by a positive-feedback mechanism.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-28476.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. H. Cox, Dept. of Physiology, Univ. of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085 (E-mail: rcox{at}mail.med.upenn.edu).

Received 3 February 1999; accepted in final form 29 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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