INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IT HAS LONG BEEN ESTABLISHED that the resting membrane potential (RMP) of vascular smooth muscle cells (SMCs) depends primarily on the gradient for potassium ions across the plasma membrane (13, 14). An inwardly rectifying K⁺ (K_IR) conductance in smooth muscle was first demonstrated by Edwards and coworkers (9), who demonstrated a K⁺-dependent inward current that rectified at potentials positive to the potassium equilibrium potential (E_K) (8, 9). However, because of the syncytial nature of smooth muscle tissue, recording of the K_IR current was not possible until single-cell isolation techniques were adopted. The first demonstration of K_IR currents in smooth muscle with the patch-clamp technique was by Quayle and coworkers (28), who characterized these currents in rat cerebral arteries. K_IR currents have since been identified in rat hepatic, coronary, and mesenteric vascular SMCs (2, 10, 17, 29).

Although K_IR currents are present in many vascular SMCs, they are not as prevalent as other K⁺ current types, like the large conductance Ca²⁺-activated K⁺ channel. Indeed, early studies demonstrated variability in smooth muscle of different vascular regions (8, 9) and thereby served to orient investigators searching for K_IR channels to SMCs of specific vessels when patch-clamp techniques were established (28). Subsequent investigations revealed that K_IR current is present in higher density in small-diameter arteries compared with larger, conduit vessels (27). This distribution suggests a fundamental diversity of K_IR channel expression in smooth muscle and is believed to contribute to functional differences between these types of vessels. However, it is uncertain whether K_IR expression can be dynamically regulated in a given population of SMCs.

To determine whether K_IR expression in vascular smooth muscle is related to cell differentiation, we characterized K_IR current in HITB5 SMCs, a human internal thoracic artery SMC clone. The HITB5 SMCs have the ability to convert between the contractile and proliferative phenotypes in culture (19, 20) and have allowed us to demonstrate (15) critical differences between these phenotypes, in terms of both contractile ability and ion channel complement. Here, we demonstrate Kir2.1 and Kir2.2 gene transcripts in human vascular SMCs as well as evidence that K_IR channels are upregulated in proliferative compared with contractile cells. The phenotype-dependent plasticity of K_IR expression revealed here indicates that these channels are subject to dynamic physiological regulation and may contribute to vascular remodeling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

HITB5 SMCs and electrophysiology. All studies were carried out with HITB5 SMCs, which can convert between a motile, proliferative phenotype and a contractile phenotype on withdrawal of serum from the medium (20). Cells studied were from subcultures 15-20 after cloning. The proliferative phenotype was attained by culturing cells in M199 with 10% FBS. The contractile phenotype was generated by culturing cells in M199 with no added serum. The electrophysiological properties of these cells were studied 72 h after serum withdrawal, a time established to yield contractile cells with increased expression of contractile-specific muscle proteins (20). For electrophysiological studies, cells were lifted from culture dishes with 0.25% trypsin-0.1 mM EDTA (GIBCO) and transferred to a 1-ml bath perfusion chamber. Cells were allowed to settle and adhere before being perfused with bath solution at a rate of 1-3 ml/min. The chamber was mounted on the stage of a Nikon inverted microscope. For whole cell recordings, the nystatin perforated-patch technique was used, with the electrode solution containing 250 µg/ml nystatin, as previously described (15).

RNA isolation and RT-PCR. Confluent SMCs were washed twice in cold PBS, and total RNA was extracted with TRIzol reagent (BRL/Life Technologies). One microgram of the total RNA was reverse transcribed into cDNA with Superscript II transcriptase (BRL/Life Technologies) and oligo d(T) primers. The cDNA was then subjected to PCR amplification. Primers for Kir2.1 were designed based on published human cardiac inward rectifier potassium channel Kir2.1 sequence (GenBank accession no. U16861) and spanned a 600-bp cDNA fragment. For Kir2.1, primers were forward 5'-AAGTCCACACCCGACAACAGTG-3' and reverse 5'-TTCTGGATTTGAGGAGCTGTGC-3'; for Kir2.2 (GenBank accession no. XM015523), forward 5'-CGCACTTCCACAAGACCTATG-3' and reverse 5'-AAAACCAGCAAGGAGCACCG-3', expected product size of 648 bp; and for Kir2.3 (GenBank accession no. XM009984), forward 5'-TTTGAGCCTGTGGTCTTCG-3' and reverse 5'-TCCCCCAGTCTTTGAAACC-3', expected product size of 531 bp. PCR was carried out for 35 cycles with denaturation at 94°C for 45 s, annealing at 54°C for 45 s, and extension at 72°C for 1.5 min. PCR product was resolved on 2% agarose gel and visualized by ethidium bromide staining. Single bands of the appropriate sizes were excised from gel, purified with a QIAquick gel extraction kit (Qiagen), and sequenced to confirm identity of the amplicons.

Western blot analysis for Kir2.1. HITB5 SMCs cultured in M199 supplemented with 10% FBS or serum starved in M199 for 3 days were used for Western blot analysis for Kir2.1 protein as described previously (20). Total cellular protein content was quantified by the Lowry assay (Bio-Rad). Equal amounts of protein (20 µg) were loaded and run on 10% SDS-PAGE gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk overnight at 4°C and then incubated with rabbit anti-human Kir2.1 antibody (1:1,000 dilution; Alomone Labs) for 1 h at room temperature. Bound primary antibody was detected with horseradish peroxidase-conjugated sheep anti-rabbit IgG F(ab')₂ (1:5,000 dilution; Amersham Pharmacia Biotech) for 1 h at room temperature, and blots were developed with SuperSignal ECL detection reagents (Pierce) before quantitation on a Bio-Rad GS-700 imaging densitometer.

DNA replication. To assess DNA synthesis rates, SMCs were pulse labeled with [³H]thymidine (20). Briefly, HITB5 cells were seeded in 96-well plates at 5 × 10³ cells/cm². Cells were incubated with [³H]thymidine for 8 h (10 µCi/ml, 71 Ci/mmol; ICN) before being transferred onto glass fiber filters with an automated cell harvester (Tomtec). Incorporated [³H]thymidine was measured with a Trilax 1450 MicroBeta counter (Wallac). We monitored cell viability on the basis of inspection of cell morphology and only studied concentrations of Ba²⁺ at which there was no evidence for a toxic effect, as shown by rounding of the cell borders, increased spacing between cells, granularity of the cytoplasm, debris in the media, or any decline in cell confluence.

Solutions. The Na⁺-HEPES bath solution used for perforated-patch recording contained (in mM) 130 NaCl, 5 KCl, 20 HEPES, 10 D-glucose, 1 CaCl₂, and 1 MgCl₂ (pH set to 7.4 with NaOH). The electrode solution used for perforated-patch recording contained (in mM) 140 KCl, 0.4 CaCl₂, 1 MgCl₂, 20 HEPES, and 1 EGTA (pH set to 7.2 with KOH). The concentration of K⁺ in the bathing solution was raised by substitution of KCl for NaCl. For cell-attached patch recording, the bath and electrode solution was composed of (in mM) 135 KCl, 20 HEPES, 10 D-glucose, 1 MgCl₂, and 1 CaCl₂ (pH set to 7.4 with KOH).

Statistical analysis. Values are provided as means ± SE, with sample sizes (n) indicating the number of cells studied. For patch-clamp experiments, only one patch was obtained per cell. Statistical comparisons were made with ANOVA or Student's t-test, with P < 0.05 considered to indicate significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Inward current in human vascular SMCs. Whole cell currents were recorded from human SMCs with the perforated-patch technique. Hyperpolarizing the membrane from a holding potential of 60 mV elicited an inward current that peaked and declined slightly with prolonged depolarization (Fig. 1). The inward current demonstrated strong rectification, with little outward current when the membrane was stepped to more positive potentials (Fig. 1A). As this inward current represented the predominant current evident over the range of resting potentials, we sought to characterize it in detail.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Hyperpolarization elicits Ba2+-sensitive inwardly rectifying current in human vascular smooth muscle cells (SMCs). A: representative currents recorded from a proliferative cell bathed in standard solution containing 5 mM extracellular K+ concentration ([K+]o) (84 pF). Hyperpolarization from a holding potential of 60 mV elicited large inward currents, whereas depolarization evoked only small outward current at positive potentials. Dotted line represents the zero-current level in this and subsequent records. B: inward current was elicited with voltage commands from 60 to 120 mV, whereupon Ba2+ (5 and 50 µM) caused a time-dependent inhibition. C: current-voltage relationships were assessed with voltage-ramp protocols. Ba2+ had no effect on outward current at positive potentials but resulted in a concentration-dependent inhibition of the inwardly rectifying K+ (KIR) current at more negative potentials. The Ba2+-sensitive current from C is expanded in the inset, determined as the difference between control current and current remaining after inhibition with 50 µM Ba2+ (I). Isolation of the Ba2+-sensitive current clearly reveals outward current between 25 and 70 mV. D: concentration dependence of Ba2+ blockade was determined as size of the current at 120 mV relative to control, when averaged between 600 and 700 ms after initiation of hyperpolarization. Each point represents mean ± SE fractional current from 3 or 4 cells, and the curve is best fit of the data, revealing half-maximal blockade at 10 µM Ba2+. All currents were recorded from proliferative cells.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Dependence of the inwardly rectifying current on [K+]o. A: current-voltage relationship was assessed with voltage-ramp commands. Cells were held at 60 mV and commanded from 120 to 50 mV at 370 mV/s. Increasing [K+]o from 5 to 20, 50, and 135 mM resulted in a positive shift of the current-voltage relationship. All currents were recorded from the same proliferative myocyte. Cell capacitance was 200 pF. B: reversal potential was determined from 5 cells by extrapolating the linear current at negative potentials to zero current. Solid line represents linear fit of reversal potentials, with a slope of 53 mV per 10-fold change of [K+]o. C: conductance of the inwardly rectifying current was determined from currents recorded in A. Conductance declined at more positive potentials and was dependent on [K+]o, demonstrating inward rectification. Traces in A were corrected for leak by subtraction of current remaining in the presence of 50 µM Ba2+ (see Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Single-channel characteristics of KIR current. Currents were recorded in the cell-attached patch configuration from proliferative cells bathed in 135 mM K+ solution. A: representative current traces from patches held at 120 mV. Single-channel events of ~3 pA were evident under control conditions (left; n = 7). Currents were abolished when 50 µM Ba2+ was added to the pipette solution, with only small downward deflections remaining (right; n = 7). B: current-voltage relationship of inwardly rectifying channel. Patches were commanded with a ramp protocol from 50 to 120 mV over 700 ms. Two representative traces are superimposed, demonstrating one or two channel openings at negative potentials and rectification as voltage approaches 0 mV. C: current amplitude was determined at various potentials with voltage step commands. Each point represents mean ± SE current (where visible as larger than symbol) for 3 or 4 cells. Single-channel conductance was 31 pS in 135 mM [K+]o, as determined by the slope of the best fit line.

Inward rectifier current density increases in proliferative phenotype. In our previous investigation of the currents in HITB5 SMCs (15), we documented differences in current activation between contractile and proliferative cells. Ca²⁺-activated Cl currents were found only in contractile cells, indicating plasticity of channel expression. A trend toward greater K_IR current in the proliferative phenotype was also observed, which we have examined here in detail. Indeed, K_IR current was generally larger in proliferative compared with contractile cells (Fig. 4A). To determine whether the amplitude was a reflection of cell size, we measured cell capacitance by off-line digital integration of capacitative currents. Both cell size and K_IR current amplitude (as measured at 120 mV) demonstrated greater variability in proliferative compared with contractile cells (Fig. 4B), with mean values of current amplitude at 120 mV of 999 ± 200 pA in proliferative cells compared with 129 ± 26 pA in the contractile phenotype. Cell size showed a similar trend, with proliferative cells demonstrating greater variability and an average capacitance of 231 ± 46 pF, whereas contractile cells were smaller with 124 ± 25 pF. When current was corrected for cell size, the proliferative phenotype demonstrated significantly larger K_IR current density (4.5 ± 0.9 pA/pF compared with 1.4 ± 0.3 pA/pF in contractile cells, P < 0.001; n = 25 for each). These findings therefore identify differences in K_IR current activity between SMC phenotypes.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Comparison of KIR current in proliferative and contractile SMCs. A: representative current traces from cells held at 60 mV and hyperpolarized to 120, 100, 80, and 60 mV with step commands. B: distribution of current at 120 mV cell capacitance demonstrates variability in cell size and current amplitude in proliferative cells (n = 25 for each). Average cell capacitance was significantly greater in proliferative compared with contractile cells. Average current density (determined as pA/pF at 120 mV) was significantly larger in cells of the proliferative phenotype (see text).

Identification of Kir2.1 and Kir2.2 mRNA in human vascular SMCs. Whole cell and single-channel experiments provided evidence for a strongly rectifying K⁺-selective channel with characteristics of channels of the Kir2 family, of which Kir2.1 has been previously identified in smooth muscle (2, 5). To investigate the molecular identity of the channels contributing to the inward rectifier currents in human vascular SMCs, we isolated and reverse transcribed total RNA from proliferative HITB5 SMCs. This clonal cell line provided a tremendous opportunity to identify mRNA transcripts without contamination from other cell types. Amplifying smooth muscle cDNA with primers specific for Kir2.1 and Kir2.2 revealed single bands of the expected size (Fig. 5A). We also carried out positive controls for each set of primers with human brain cDNA. In contrast, there was no evidence of Kir2.3 in smooth muscle, although a band was present in the brain control (Fig. 5A, right). Similar results were obtained in three independent experiments. Sequencing of the amplicons confirmed homology to the published gene sequences. RNA that had not undergone reverse transcription was also subjected to the amplification procedure, and no bands were evident (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 5.   KIR gene transcripts in human vascular smooth muscle. A: RNA was extracted from proliferative SMCs and reverse transcribed as described in MATERIALS AND METHODS. cDNA was amplified with primers designed for Kir2.1 (600-bp region), Kir2.2 (648-bp region), and Kir2.3 (531-bp region). Staining with ethidium bromide after PCR revealed a single band of expected size for all channel types in the brain, but only Kir2.1 and Kir2.2 were evident in smooth muscle. PCR products were subsequently sequenced and confirmed to be the correct channel sequence. For control experiments, PCR was carried out with specific primers on RNA samples that had not undergone reverse transcription (not shown). B: Western blot analysis of Kir2.1 channel protein. Equal amounts of protein isolated from proliferative and contractile cells were run on SDS-PAGE gel and then incubated with rabbit anti-human Kir2.1 antibody. Denser band indicates that proliferative cells expressed Kir2.1 in greater quantity. MW, molecular weight. C: average band density for 3 replicate experiments confirmed that Kir2.1 expression is significantly greater in proliferative than contractile cells (*P < 0.05).

K_IR contributes to regulation of membrane potential. We next investigated the contribution of the inwardly rectifying K⁺ current to the membrane potential of both contractile and proliferative cells. Figure 6A shows a representative trace from a proliferative cell recorded in current-clamp configuration. Current was injected at 5-s intervals to evaluate membrane conductance. External Ba²⁺ caused rapid and reversible depolarization, indicating that K_IR contributed to setting the RMP. Furthermore, increases in [K⁺]_o from 5 to 135 mM resulted in progressive depolarization, close to that predicted by the Nernst equation. Voltage deflections recorded during current injection increased in amplitude during Ba²⁺ application, as expected during channel inhibition. In contrast, conductance increased with higher [K⁺]_o, resulting in smaller voltage deflections (Fig. 6). On average, proliferative cells demonstrated a more negative resting potential compared with contractile cells (71 ± 2 mV for 9 proliferative cells, 35 ± 4 mV for 5 contractile cells; P < 0.001; Fig. 6B). Ba²⁺ (50 or 100 µM) depolarized cells of both phenotypes, with proliferative cells dropping to 50 ± 6 mV (P < 0.005; n = 6) and contractile cells to 25 ± 3 mV (P < 0.05; n = 3). These data demonstrate that the Ba²⁺-sensitive current contributes to setting the RMP of each phenotype. Moreover, the more hyperpolarized RMP of the proliferative cells may reflect the increased Kir2.1 channel expression shown above.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Functional role of Kir in setting the resting membrane potential of human vascular myocytes. A: membrane potential (Vm) recorded from a representative proliferative cell. Ba2+ (100 µM) caused prompt depolarization, with recovery on washout. Hyperpolarizing current steps (50 pA, 0.5 s) were applied every 5 s to assess input resistance. Increasing [K+]o elicited progressive membrane depolarization. Cell capacitance was 128 pF. B: proliferative cells demonstrated a more negative resting potential compared with contractile cells, and Ba2+ (50 or 100 µM) elicited depolarization of both cell types (* P < 0.05 for all comparisons).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Kir is responsible for K+-induced hyperpolarization in contractile human vascular SMCs. A: representative voltage trace from a contractile cell studied in current clamp. Increasing [K+]o from 5 to 12 mM caused prompt hyperpolarization, with membrane potential returning to basal levels after washout (n = 5). B: current-voltage relationship of cell in A demonstrating KIR current that was augmented by the increased [K+]o. C: expanded traces from B reveal a shift in the reversal potential from 37 mV in 5 mM [K+]o to 46 mV in 12 mM [K+]o.

Ba²+ inhibits [³H]thymidine incorporation in proliferative vascular SMCs. Proliferative HITB5 cells demonstrated greater Kir2.1 expression than contractile cells. Furthermore, K_IR activity contributed to the more hyperpolarized RMP of the proliferative compared with contractile cells. Therefore, we chose to investigate the contribution of K_IR current to cell proliferation. Proliferative SMCs were pulse labeled with [³H]thymidine, and incorporation was assessed. The addition of Ba²⁺ decreased [³H]thymidine incorporation in a concentration-dependent manner (Fig. 8) suggestive of a decrease in cell proliferation. DNA synthesis was reduced to 50% in the presence of 1 mM Ba²⁺, indicating a decrease in potency compared with channel block at 120 mV, which is in keeping with the steep voltage dependence of Ba²⁺ blockade (29).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Ba2+ decreases [3H]thymidine incorporation in proliferative vascular SMCs. Thymidine incorporation was assessed by pulsing cells with 10 µCi/ml [3H]thymidine for 8 h. Extracellular Ba2+ caused a dose-dependent decrease in thymidine incorporation. Results are expressed as means ± SE of 12 samples at each concentration (* significant difference of P < 0.05, as determined by ANOVA and Student-Newman-Keuls post hoc test). Experiments were duplicated with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we demonstrated phenotype-dependent plasticity of K_IR current and channel expression in human vascular SMCs. Patch-clamp recording provided evidence for differential activity of this current in the HITB5 clone, with proliferative cells exhibiting greater current than contractile cells. RT-PCR revealed transcripts for Kir2.1 and Kir2.2. Western blot analysis for Kir2.1 revealed increased Kir2.1 protein in proliferative cells, consistent with the regulated expression of this channel. K_IR current also contributed to setting the RMP, with proliferative cells showing a more hyperpolarized resting potential compared with contractile cells. K_IR current may participate in the regulation of vascular SMC proliferation, as extracellular Ba²⁺ inhibits [³H]thymidine incorporation in proliferative cells.

K_IR current was identified in HITB5 SMCs by its inward rectification, sensitivity to external Ba²⁺, and K⁺ selectivity. These characteristics resemble those previously demonstrated for K_IR currents in SMCs from cerebral and coronary arteries (2, 27-29) and are indicative of a channel from the Kir2 family (23). Although the inward component of the K_IR current has been used for identification and characterization in most studies on vascular myocytes, the physiologically significant outward component of the current has until now only been clearly shown in cell types other than smooth muscle (16) and oocytes expressing Kir2.1 (18). The robust expression of this channel in the proliferative phenotype allowed us to demonstrate the outward current for the first time in human SMCs.

The K⁺ selectivity of the K_IR current in the present study was evaluated by increasing [K⁺]_o, and the reversal potential was shifted by a 53-mV/10-fold change in [K⁺]_o, similar to previous reports (16, 28, 29) and consistent with the value predicted by the Nernst equation for a K⁺-selective channel. Furthermore, the conductance increased with a rise in [K⁺]_o, further implicating a channel of the Kir2 family. These channels are also characterized by their sensitivity to external Ba²⁺ in the low micromolar range, similar to previous reports in vascular SMCs (2, 28, 29). Many earlier studies of K_IR in smooth muscle were carried out with elevated [K⁺]_o to enhance the inwardly rectifying current and increase the driving force. When the blocker data from Bradley and coworkers (2) are considered at 40 mV (approximately the driving force used in the present study), the half-blockade by Ba²⁺ occurred at 7.2 µM, close to what we observed. The single-channel properties of the K_IR current were also investigated. The K_IR current activity in the proliferative phenotype was robust, allowing us to demonstrate current rectification at the single-channel level as well as blockade by Ba²⁺. The unitary conductance of 25-30 pS we found is in the range reported for Kir2.1 (22 pS) and Kir2.2 (34 pS) channels in other cell types (23). This is the first description of single-channel K_IR currents in vascular SMCs, and it further supports the conclusion that this channel is of the Kir2 family.

RT-PCR analysis of total cell mRNA revealed mRNA transcripts for Kir2.1 and Kir2.2, but not Kir2.3, in human vascular myocytes. Electrophysiological reports suggested that the current derived from a member of the Kir2 family, and indeed Kir2.1 mRNA has been identified in the rat basilar artery (2, 5) as well as in the coronary and mesenteric artery (2). It is of interest that immunocytochemical studies indicate the presence of all three Kir channel types in rat cerebral and basilar arteries, although expression of Kir2.3 was reported to be weak compared with Kir2.1 and Kir2.2 (31).

Interestingly, smooth muscle K_IR channels have so far only been investigated in the contractile phenotype. Here we compare the expression and activity of Kir2.1 in both the contractile and proliferative phenotypes of a vascular SMC cell clone (20). These cells bidirectionally switch phenotype, allowing for the comparison of phenotypes while avoiding complications arising from extended time in culture (12, 24). This enabled us to demonstrate that loss of the contractile phenotype need not be accompanied by a concurrent loss of large conductance Ca²⁺-activated K⁺ channels in SMCs (15), as suggested earlier (22). However, Ca²⁺-activated Cl current activity was observed exclusively in contractile cells, indicating plasticity of Cl channel expression in vascular SMCs (15). Here we have identified another example of phenotype-dependent expression of ion channels in human SMCs. Patch-clamp electrophysiology and Western blot analysis demonstrated greater K_IR current density and protein in the proliferative compared with the contractile phenotype, indicating that the expression of this channel can be dynamically regulated within a defined population of vascular SMCs.

The involvement of K_IR channels in setting the membrane potential of contractile SMCs was investigated, as the resting potential is a key determinant of myogenic tone (5, 9). K_IR channels have also been implicated in the phenomenon of K⁺-induced hyperpolarization whereby small increases in [K⁺]_o cause vasodilation in vitro (3, 4, 10, 17, 21, 27) and in vivo (5). A study of Kir2.1 knockout mice confirmed this role for K_IR, as these animals no longer demonstrate vasodilation in response to increases in [K⁺]_o (33). In the present study, we demonstrate for the first time K⁺-induced hyperpolarization at the level of the individual vascular SMC cell with the current-clamp technique. Contractile cells that had an average membrane potential of approximately 35 mV hyperpolarized in response to a small elevation in [K⁺]_o. Moreover, those cells that did not show inward rectifier current in voltage clamp also did not hyperpolarize, implicating K_IR in this process.

In contrast, hyperpolarization in response to small increases in [K⁺]_o was not observed in SMCs of the proliferative phenotype. This is consistent with the hyperpolarized RMP of these cells; on average, proliferative cells had a RMP of approximately 70 mV. Increases in [K⁺]_o only caused membrane depolarization due to the positive shift in E_K. However, proliferative cells were dependent on K_IR for the determination of the membrane potential, suggesting that the inward rectifier current, and the resulting hyperpolarization, serves an important functional role. Indeed, [³H]thymidine incorporation was decreased by addition of the K_IR channel blocker Ba²⁺, suggesting that K_IR may participate in the regulation of cell proliferation.

Membrane potential is believed to be a vital determinant of vascular SMC proliferation, as depolarized potentials will allow for greater Ca²⁺ influx, a known mitogenic signal (1), through voltage-dependent L-type Ca²⁺ channels (26). Evidence to support this idea comes from studies documenting the regulation of several classes of K⁺ currents, including voltage-dependent delayed rectifier current (26) and ATP-sensitive K⁺ channels (6), where in both cases proliferating cells exhibit reduced K⁺ current density. These findings raise the possibility that different mechanisms may regulate the growth of smooth muscle cells originating in different tissues. However, Curtis and Scholfield (7) demonstrated the presence of a nifedipine-sensitive but non-L-type Ca²⁺ influx pathway in arteriolar SMCs and concluded that a hyperpolarized membrane potential would maximize Ca²⁺ influx by increasing the Ca²⁺ driving force, dogma generally reserved for nonexcitable cells. K_IR currents play a critical role in proliferation of other cell types, possibly by affecting the RMP. Schlichter and colleagues (30) showed that K_IR currents are necessary for colony stimulating factor-1-induced proliferation of microglial cells. Also, Vaur and associates (32) observed depolarization and decreased proliferation with blockade of K_IR current in a pituitary cell line.

Further evidence for the role of Kir2.1 in cell proliferation and development comes from genetic disruption of Kir2.1. Zaritsky and coworkers (33) suggested that the lethal cleft palate observed in Kir2.1-deficient mice is due to a breakdown in cell signaling, proliferation, or differentiation of various cell types. These conclusions are supported by the phenotype of patients suffering from Andersen syndrome, which was recently attributed to mutations in Kir2.1 and is characterized by cardiac arrythmias, skeletal muscle myotonia, and dysmorphia (25). Plaster and coworkers (25) recognized that the cleft palate of the Kir2.1 knockout mouse corresponded to the dysmorphic features seen with Andersen syndrome and concluded that Kir2.1 plays a critical role in development. Furthermore, decreasing Kir2.1 expression has been shown to retard skeletal muscle development in vitro (11). These findings are consistent with our data and suggest that Kir2.1 expression may be a key factor in vascular SMC proliferation.

In conclusion, we demonstrated phenotype-dependent plasticity of K_IR expression and activity in human vascular SMCs. These channels contribute to both the regulation of myogenic tone and the rate of SMC [³H]thymidine incorporation. The dynamically regulated expression of K_IR, including Kir2.1, may play a critical role in physiological processes such as vascular remodeling.