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Genetic ablation of caveolin-1 modifies Ca²⁺ spark coupling in murine arterial smooth muscle cells

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 21 November 2005 ; accepted in final form 17 January 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
L-type, voltage-dependent calcium (Ca²⁺) channels, ryanodine-sensitive Ca²⁺ release (RyR) channels, and large-conductance Ca²⁺-activated potassium (K_Ca) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters Ca²⁺ spark to K_Ca channel coupling and Ca²⁺ spark regulation by voltage-dependent Ca²⁺ channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control (cav-1^+/+) cells but were not observed in cav-1-deficient (cav-1^–/–) cells. Ca²⁺ spark and transient K_Ca current frequency were approximately twofold higher in cav-1^–/– than in cav-1^+/+ cells. Although voltage-dependent Ca²⁺ current density was similar in cav-1^+/+ and cav-1^–/– cells, diltiazem and Cd²⁺, voltage-dependent Ca²⁺ channel blockers, reduced transient K_Ca current frequency to 55% of control in cav-1^+/+ cells but did not alter transient K_Ca current frequency in cav-1^–/– cells. Furthermore, although K_Ca channel density was elevated in cav-1^–/– cells, transient K_Ca current amplitude was similar to that in cav-1^+/+ cells. Higher Ca²⁺ spark frequency in cav-1^–/– cells was not due to elevated intracellular Ca²⁺ concentration, sarcoplasmic reticulum Ca²⁺ load, or nitric oxide synthase activity. Similarly, Ca²⁺ spark amplitude and spread, the percentage of Ca²⁺ sparks that activated a transient K_Ca current, the amplitude relationship between sparks and transient K_Ca currents, and K_Ca channel conductance and apparent Ca²⁺ sensitivity were similar in cav-1^+/+ and cav-1^–/– cells. In summary, cav-1 ablation elevates Ca²⁺ spark and transient K_Ca current frequency, attenuates the coupling relationship between voltage-dependent Ca²⁺ channels and RyR channels that generate Ca²⁺ sparks, and elevates K_Ca channel density but does not alter transient K_Ca current activation by Ca²⁺ sparks. These findings indicate that cav-1 is required for physiological Ca²⁺ spark and transient K_Ca current regulation in cerebral artery smooth muscle cells.

ryanodine-sensitive Ca²⁺ release channel; large-conductance Ca²⁺-activated potassium channel; caveolae; voltage-dependent Ca²⁺ channel

Smooth muscle cells generate several different modes of Ca²⁺ signaling (15, 20). The global intracellular Ca²⁺ concentration ([Ca²⁺]_i) arises because of extracellular Ca²⁺ influx and intracellular Ca²⁺ release. An elevation in global [Ca²⁺]_i stimulates contraction, whereas a reduction in global [Ca²⁺]_i results in relaxation. Localized [Ca²⁺]_i transients, termed "Ca²⁺ sparks," also occur in smooth muscle cells (20, 27). Ca²⁺ sparks occur due to the opening of several ryanodine-sensitive Ca²⁺ release (RyR) channels on the sarcoplasmic reticulum (SR) (15, 27). In smooth muscle cells, a Ca²⁺ spark does not directly elevate global Ca²⁺ but activates a number of nearby large-conductance Ca²⁺-activated potassium (K_Ca) channels, resulting in a transient K_Ca current. Membrane hyperpolarization induced by asynchronous transient K_Ca currents reduces voltage-dependent Ca²⁺ channel activity, leading to a decrease in global [Ca²⁺]_i and relaxation. An elevation in [Ca²⁺]_i activates Ca²⁺ sparks and transient K_Ca currents, forming a negative-feedback loop that limits Ca²⁺ entry through voltage-dependent Ca²⁺ channels (15, 27). Thus voltage-dependent Ca²⁺ channels, RyR channels, and K_Ca channels comprise a functional unit that regulates smooth muscle contractility (17). The differential regulation of contractility by local and global Ca²⁺ signaling is also effective because of differences in the frequency and amplitude of the Ca²⁺ signals and the Ca²⁺ sensitivities of the downstream targets for each signal mode (15). For local Ca²⁺ signaling mechanisms to operate, downstream targets must also be located in the proximity of the Ca²⁺ source. In smooth muscle cells, whether membrane proteins maintain spatial organization of voltage-dependent Ca²⁺ channels, K_Ca channels, and RyR channels to permit Ca²⁺ signaling between these proteins is unclear.

Compartmentalization of signaling molecules can occur in small, cholesterol-enriched, flask-shaped membrane invaginations termed "caveolae" (11, 13, 34, 39). Caveolins, of which three isoforms have been cloned (cav 1–3), are structural components required for caveolae formation (11, 13, 34, 39). Although all three caveolins have been identified in vascular smooth muscle cells, caveolin-1 is the primary isoform (9, 18, 28, 34, 39). Supporting a role for caveolae in the regulation of arterial smooth muscle local Ca²⁺ signaling is evidence that acute cholesterol depletion with dextrin inhibits Ca²⁺ sparks (23). Similarly, transient K_Ca current frequency is reduced in cerebral artery smooth muscle cells of cav-1-deficient (cav-1^–/–) mice, when compared with wild-type controls (10). In ureter smooth muscle cells, K_Ca channels are found in buoyant membrane fractions, and in cultured myometrial cells K_Ca channels are localized to caveolae (1, 3). Thus cav-1 and caveolae may regulate Ca²⁺ sparks and K_Ca channels in smooth muscle.

Here, the communication between voltage-dependent Ca²⁺ channels and RyR channels that generate Ca²⁺ sparks and signaling between Ca²⁺ sparks and K_Ca channels was investigated in cerebral artery smooth muscle cells of cav-1^+/+ and cav-1^–/– mice. Data indicate that cav-1 ablation abolishes caveolae, elevates Ca²⁺ spark frequency, and attenuates Ca²⁺ spark regulation by voltage-dependent Ca²⁺ channels. In contrast, transient K_Ca current activation by Ca²⁺ sparks is unaltered in cav-1^–/– cells, even though there is an increase in K_Ca channel density.

	MATERIALS AND METHODS

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Tissue preparation. Animal procedures used were reviewed and approved by the Animal Care and Use Committee policies at the University of Tennessee. Age-matched (4–6 wk) cav-1^–/– mice (Cav1^tm1Mls, stock no. 004585; see Ref. 33) or wild-type control (cav-1^+/+) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were killed by peritoneal injection of a pentobarbital sodium overdose (130 mg/kg). The brain was removed and placed into ice-cold (4°C) physiological saline solution (PSS) containing (in mM) 112 NaCl, 4.8 KCl, 26 NaHCO₃, 1.8 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, and 10 glucose and gassed with 74% N₂-21% O₂-5% CO₂ (pH 7.4). Posterior cerebral, cerebellar, and middle cerebral arteries (150 µm in diameter) were removed, cleaned of connective tissue, and maintained in ice-cold Ca²⁺-free buffer (solution A) containing (in mM) 55 NaCl, 80 Na glutamate, 5.6 KCl, 2 MgCl₂, 10 HEPES, and 10 glucose (pH 7.3 with NaOH). For single cell isolation, smooth muscle cells were dissociated from cerebral arteries with the use of enzymes, as previously described (14).

Electron microscopy. The brain was fixed in PSS containing 2.5% glutaraldehyde for 1 h. Cerebral arteries were dissected from the fixed brain, cleaned of connective tissue, and postfixed in 1% osmium tetroxide in PSS for 4 h. Arteries were then rinsed briefly in deionized water and en bloc stained with 2% uranyl acetate in 0.85% sodium chloride overnight at 4°C. Arteries were dehydrated in graded solutions of ethanol, from 30% through 100% for 1 h each, infiltrated with 50% Spurr in 100% ethanol overnight at room temperature, 100% Spurr over an 8-h period involving at least three changes of Spurr, and then cured at 60°C for 2 days. Transverse sections (75 nm) were cut with a Reichert Ultracut E microtome and poststained with uranyl acetate and lead citrate. Cells were observed and photographed with a JEOL 2000EX TEM located in the Electron Microscope Facility at the University of Tennessee Health Science Center.

Patch-clamp electrophysiology. Potassium currents were measured by using the conventional whole cell, perforated-patch or inside-out patch-clamp configurations with an Axopatch 200B amplifier and Clampex 8.2 (Axon Instruments, Union City, CA). For transient K_Ca current measurement, the bath solution (solution B) contained (in mM) 134 NaCl, 6 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4 with NaOH), and the pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES, and 0.05 EGTA (pH 7.2 with KOH). Amphotericin B (Sigma) was dissolved in DMSO and diluted into the pipette solution to give a final concentration of 200 µg/ml. For whole cell K⁺ current measurement, the bath solution was solution B and the pipette solution contained (in mM) 135 KCl, 5 EGTA, 1.0 BAPTA, 3.5 MgCl₂, 2.5 Na₂ATP, and 10 HEPES (pH 7.2 with KOH). For inside-out patch-clamp experiments to measure single K_Ca channel currents, the pipette and bath solution both contained (in mM) 140 KCl, 10 HEPES, 5 EGTA, and 1.6 1-N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (pH 7.2 with KOH). MgCl₂ and CaCl₂ were supplemented to bath solution to provide 1 mM free Mg²⁺ and the required free Ca²⁺ concentration (1–100 µM). Free Ca²⁺ concentration in the pipette solution was 10 µM. Free Ca²⁺ concentrations were measured by using a Ca²⁺-sensitive and reference electrode (Corning). For voltage-dependent Ba²⁺ current measurements, the pipette solution contained (in mM) 125 CsCl, 20 tetraethylammonium chloride (TEACl), 1.0 MgCl₂, 0.5 EGTA, 10 HEPES, and 10 glucose (pH 7.2 with CsOH), and the bath solution contained (in mM) 60 NaCl, 60 TEACl, 1.0 MgCl₂, 20 BaCl₂, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). Pipette resistance was measured by applying a 5-mV pulse using the seal test function of pClamp 8.2. Transient K_Ca currents were measured at a steady membrane potential of –40 mV. Voltage-dependent Ba²⁺ currents were activated from a holding potential of –80 mV by applying 300-ms voltage steps to voltages between –50 and +60 mV in increments of 10 mV. Whole cell K⁺ (I_k) currents were activated from a holding potential of –80 mV by applying 250-ms voltage steps to voltages between –70 and +80 mV in 10-mV increments. In inside-out patches, single K_Ca channel currents were measured at steady voltages of –40 or +40 mV. Transient K_Ca currents were filtered at 1 kHz and digitized at 5 kHz. Voltage-dependent Ba²⁺ currents were filtered at 1 kHz and digitized at 4 kHz. Other current measurements were filtered at 2 kHz and digitized at 10 kHz. Transient K_Ca current analysis was performed off-line using methodology described elsewhere (6). A transient K_Ca current was defined as the simultaneous opening of three or more K_Ca channels. Open probability (P_o) was calculated from the following equation: P_o = (t_ii)/nT, where t_i is the time at each channel level i, n is the number of channels in the patch, and T is the total time of analysis. The total number of K_Ca channels in an inside-out patch was determined at a voltage of +40 mV with 100 µM free Ca²⁺ in the bath solution. In each patch under each condition, at least 5 min of continuous data were analyzed to calculate transient K_Ca current frequency and amplitude, and 2–5 min were analyzed to determine single K_Ca channel open probability.

Confocal Ca²⁺ imaging. Isolated smooth muscle cells were incubated in solution A containing fluo-4 AM (10 µM) for 20 min at room temperature, followed by a 30-min wash to allow indicator deesterification. Smooth muscle cells were imaged with the use of a Noran Oz laser scanning confocal microscope (Noran Instruments, Middleton, WI) and a x60 water immersion objective (1.2 numerical aperture) attached to a Nikon TE300 microscope. Fluo-4 was excited by using the 488 nm line of a krypton-argon laser, and emitted light >500 nm was captured. Images (56.3 x 52.8 µm) were recorded every 8.3 ms (120 images/s). The laser intensity used did not alter transient K_Ca current frequency or amplitude. Current and fluorescence measurements were synchronized by using a light-emitting diode placed above the recording chamber that was triggered during acquisition. Each cell was imaged for 15 s. Ca²⁺ sparks in smooth muscle cells were analyzed with the use of software kindly provided by Dr. M. T. Nelson (University of Vermont). Detection of Ca²⁺ sparks was performed by dividing an area 1.54 µm (7 pixels) x 1.54 µm (7 pixels) (i.e., 2.37 µm²) in each image (F) by a baseline (F₀) that was determined by averaging 10 images without Ca²⁺ spark activity. The entire area of each image was analyzed to detect Ca²⁺ sparks. A Ca²⁺ spark was defined as a local increase in F/F₀ that was >1.2.

Fura-2 imaging. Cerebral arteries were incubated with the ratiometric fluorescent Ca²⁺ indicator fura-2 AM (5 µM) and 0.05% pluronic F-127 for 20 min, followed by a 15-min wash. All experiments were performed using solution B (composition described in Patch-clamp electrophysiology). Fura-2 was alternately excited at 340 or 380 nm using a PC-driven hyperswitch (Ionoptix, Milton, MA). Background corrected ratios were collected every 1 s at 510 nm using a photomultiplier tube (Ionoptix). SR Ca²⁺ load ([Ca²⁺]_SR) was estimated by rapidly applying a high concentration of caffeine (10 mM), an RyR channel activator, and measuring the amplitude of [Ca²⁺]_i transients (i.e., [Ca²⁺]_i). Intracellular Ca²⁺ concentrations were calculated by using the following equation (12):

where R is the 340/380 nm ratio; R_min and R_max are the minimum and maximum ratios determined in Ca²⁺-free and saturating Ca²⁺ solutions, respectively, S_f2/S_b2 is the ratio of Ca²⁺-free to Ca²⁺-replete emissions at 380 nm excitation, and K_d is the dissociation constant for fura-2 (282 nM) (19). R_min, R_max, S_f2, and S_b2 were determined at the end of experiments and in separate experiments by increasing the Ca²⁺ permeability of smooth muscle cells with ionomycin (10 µM) and by perfusing cells with a high-Ca²⁺ (10 mM) or Ca²⁺-free (no added Ca²⁺, 5 mM EGTA) solution.

Measurement of cellular dimensions. Isolated smooth muscle cells were allowed to settle on a glass coverslip in a chamber with Ca²⁺-free, HEPES-containing patch-clamp bath solution (composition described in Patch-clamp electrophysiology). Cell images were acquired with the use of a Zeiss LSM5 confocal microscope. Cell length and width were calculated using Pascal software (Zeiss).

Statistics. Values are expressed as means ± SE. Student's t-test and Student-Newman-Keuls test were used for comparing paired or unpaired data and multiple data sets, respectively. Simultaneous Ca²⁺ spark and transient K_Ca current amplitude data were fit with a linear regression function, and the slope ± SE of each fit was compared using Student’s t-test and Graphpad Prizm (San Diego, CA). P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Caveolae are absent in cerebral artery smooth muscle cells of cav-1^–/– mice. Electron microscopy revealed that abundant caveolae were present in the sarcolemma of smooth muscle cells of small-diameter (150 µm) cerebral arteries of cav-1^+/+ mice (Fig. 1, A and B). In contrast, caveolae were not observed in cerebral artery smooth muscle cells of cav-1^–/– mice (Fig. 1, C and D). These data indicate that cav-1 is necessary for caveolae formation in smooth muscle cells of small cerebral arteries.

View larger version (140K): [in this window] [in a new window]   Fig. 1. Genetic ablation of caveolin-1 (cav-1) abolishes caveolae in cerebral artery smooth muscle cells. A: electron micrograph illustrates sarcolemma of smooth muscle cell in cerebral artery from cav-1+/+ mouse. Abundant caveolae are present within sarcolemma. B: magnification of area illustrated by black box in A showing caveolae. C: caveolae were absent in sarcolemma of cav-1–/– cerebral artery smooth muscle cells. D: magnification of area highlighted by black box in C.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Cav-1 ablation elevates transient large-conductance Ca2+-activated potassium (KCa) current frequency in cerebral artery smooth muscle cells. A: original traces illustrate transient KCa currents in a cav-1+/+ (top) and cav-1–/– (bottom) cell at –40 mV. B: mean transient KCa current frequency was higher in cav-1–/– (n = 31) when compared with cav-1+/+ (n = 35) cells. C: mean transient KCa current amplitude was similar in cav-1+/+ (n = 35) and cav-1–/– (n = 31) cells. D: mean cell capacitance of cav-1–/– cells (n = 49) was smaller than for cav-1+/+ cells (n = 55). E: thapsigargin (100 nM) abolishes transient KCa currents in a cav-1–/– cell voltage-clamped at –40 mV. Similar results were obtained in 4 cells. *P < 0.05 when compared with cav-1+/+ cells.

In arterial smooth muscle cells, transient K_Ca currents occur due to SR Ca²⁺ release (38). We sought to determine mechanisms that activate transient K_Ca currents in cav-1^–/– cells. In cav-1^–/– cells, thapsigargin (100 nM), an SR Ca²⁺ ATPase inhibitor, abolished transient K_Ca currents, indicating that these events occur due to SR Ca²⁺ release (Fig. 2E; n = 4).

Ca²⁺ spark frequency is elevated in cav-1^–/– cells when compared with cav-1^+/+ cells, but the amplitude relationship between Ca²⁺ sparks and transient K_Ca currents is similar. Elevated transient K_Ca current frequency in cav-1^–/– cells could occur because of an increase in Ca²⁺ spark frequency or an increase in the percentage of Ca²⁺ sparks that activate a transient K_Ca current (i.e., percent coupling). To investigate these possibilities and to compare Ca²⁺ spark properties in cav-1^+/+ and cav-1^–/– cells, simultaneous measurements of Ca²⁺ sparks and evoked transient K_Ca currents were obtained by performing confocal Ca²⁺ imaging in combination with patch-clamp electrophysiology.

At –40 mV, Ca²⁺ spark frequency was 1.8-fold higher in cav-1^–/– cells than in cav-1^+/+ cells (Fig. 3, A and B). In contrast, Ca²⁺ spark amplitude (Fig. 3C) and spatial spread [full width at half-maximal amplitude; cav-1^+/+, 1.82 ± 0.13 µm (n = 40); cav-1^–/–, 1.94 ± 0.11 µm (n = 59)] were similar in cav-1^+/+ and cav-1^–/– cells (P > 0.05 for each). The percentage of Ca²⁺ sparks that activated a transient K_Ca current (cav-1^+/+, 67 ± 8%; cav-1^–/–, 71 ± 4%) and the amplitude relationship between Ca²⁺ sparks and transient K_Ca currents were also similar (Fig. 3, A and D). Taken together, these data indicate that genetic ablation of cav-1^–/– elevates Ca²⁺ spark frequency, leading to an increase in transient K_Ca current frequency.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Ca2+ spark frequency is higher in cav-1–/– cells than in cav-1+/+ cells. A: simultaneous measurements of Ca2+ sparks (bottom colored traces) and transient KCa currents (top black traces) at –40 mV in a cav-1+/+ cell (left) and cav-1–/– cell (right). Inset: peak of Ca2+ spark, labeled by asterisk in A, with yellow box in image indicating region from where F/F0 trace was obtained. B: mean Ca2+ spark frequency in cav-1+/+ (n = 9) and cav-1–/– (n = 7) cells. C: mean Ca2+ spark amplitude in cav-1+/+ (n = 88) and cav-1–/– (n = 124) cells. D: amplitude correlation of Ca2+ sparks and evoked transient KCa currents in cav-1+/+ (black squares) and cav-1–/– cells (red circles) with linear fit and 95% confidence bands to each data set [slope ± SE: cav-1+/+, 14.2 ± 1.4 (r = 0.25, n = 62, P < 0.0001); cav-1–/–, 16.0 ± 1.1 (r = 0.32, n = 91, P = 0.01)]. Amplitude relationship was not significantly different in cav-1+/+ and cav-1–/– cells (P > 0.05). *P < 0.05 when compared with cav-1+/+ cells.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Intracellular Ca2+ concentration ([Ca2+]i) and sarcoplasmic reticulum Ca2+ load are similar in cav-1+/+ and cav-1–/– cerebral arteries. A: [Ca2+]i and caffeine (10 mM)-induced Ca2+ transients were measured in cav-1+/+ and cav-1–/– cerebral arteries using fura-2. B: [Ca2+]i was similar in cav-1+/+ (n = 7) and cav-1–/– (n = 9) cerebral arteries. C: caffeine-induced Ca2+ transients were similar in cav-1+/+ (n = 7) and cav-1–/– (n = 9).

N-nitro-L-arginine does not inhibit transient K_Ca currents in cav-1^+/+ or cav-1^–/– cells.

Transient K_Ca current regulation by voltage-dependent Ca²⁺ channels is abolished in cav-1^–/– cells. In smooth muscle cells, Ca²⁺ sparks are activated by Ca²⁺ entering through sarcolemma voltage-dependent Ca²⁺ channels (14, 16, 20). We investigated whether Ca²⁺ sparks are more frequent in cav-1^–/– cells because of enhanced coupling between voltage-dependent Ca²⁺ channels and RyR channels. In cav-1^+/+ cells, Cd²⁺ (250 µM), a voltage-dependent Ca²⁺ channel blocker, or diltiazem (50 µM), an L-type Ca²⁺ channel blocker, reduced transient K_Ca current frequency to 51 and 57% of control, respectively, but did not alter transient K_Ca current amplitude (Fig. 5, A–C). In contrast, Cd²⁺ and diltiazem did not alter transient K_Ca current frequency or amplitude in cav-1^–/– cells (Fig. 5, A–C). Thus Ca²⁺ spark regulation by voltage-dependent Ca²⁺ channels is abolished in cav-1^–/– cells, indicating that higher Ca²⁺ spark frequency is not through enhanced coupling between voltage-dependent Ca²⁺ channels and RyR channels.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Voltage-dependent Ca2+ channel blockers reduce transient KCa current frequency in cav-1+/+ cells but not in cav-1–/– cells. A: original traces illustrating effect of CdCl2 (250 µM) on transient KCa currents in a cav-1+/+ and cav-1–/– cell at –40 mV. B: mean effects of CdCl2 on transient KCa current frequency and amplitude in cav-1+/+ (n = 5) and cav-1–/– (n = 6) cells. C: mean effect of diltiazem (50 µM) on transient KCa currents in cav-1+/+ (n = 5) and cav-1–/– (n = 4) cells. *P < 0.05 when compared with control obtained in same cells before Cd2+ or diltiazem application; #P < 0.05 when compared with cav-1+/+ cells.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Voltage-dependent Ca2+ current density is similar in cav-1+/+ and cav-1–/– cells. A: original traces illustrating voltage-dependent Ba2+ currents (IBa) elicited by a voltage step from a holding potential of –80 to +20 mV in cav-1+/+ and cav-1–/– cerebral artery smooth muscle cells. CdCl2 (250 µM) abolished (IBa) in cav-1+/+ and cav-1–/– cells. B: current density-voltage relationship of CdCl2 (250 µM)-sensitive currents in cav-1+/+ (n = 9) and cav-1–/– (n = 9) cells. P > 0.05 for each voltage when compared with cav-1+/+.

Transient K_Ca current amplitude is dependent on K_Ca channel conductance and Ca²⁺ sensitivity (4). In excised inside-out patches, single K_Ca channel slope conductance between –40 and +40 mV [cav-1^+/+, 268 ± 3 pS (n = 9); cav-1^–/–, 260 ± 3 pS (n = 7); P > 0.05] and K_Ca channel-apparent Ca²⁺ sensitivity were similar in cav-1^+/+ and cav-1^–/– cells (Fig. 7, A and B). Transient K_Ca current amplitude also depends on the number of K_Ca channels activated by a Ca²⁺ spark. On average, inside-out patches pulled from cav-1^–/– cells contained 1.5-fold more K_Ca channels than patches obtained from cav-1^+/+ cells (Fig. 7C). This finding was not due to differences in the size of patch pipettes used for these experiments [cav-1^+/+, 28.6 ± 1.2 M (n = 17); cav-1^–/–, 27.1 ± 0.7 M (n = 22); P > 0.05]. These data suggest that sarcolemma K_Ca channel density is higher in cav-1^–/– cells than in cav-1^+/+ cells.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Single KCa channel properties in cav-1+/+ and cav-1–/– cells. A: original records illustrate single KCa channel openings in 10 µM Ca2+ at –40 and +40 mV in inside-out patches from a cav-1+/+ and cav-1–/– cell. B: at –40 and +40 mV, apparent Ca2+ sensitivity of KCa channels was similar for cav-1+/+ (n = 8–9 for each [Ca2+]) and cav-1–/– (n = 7 for each [Ca2+]) cells (P > 0.05 for each). Po, open probability. C: inside-out patches from cav-1–/– cells (n = 22) contained more KCa channels than patches from cav-1+/+ cells (n = 17). C, closed; O, open. *P < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Whole cell K+, KCa, and KV current density is elevated in cav-1–/– cells. A: original recordings of whole cell K+ currents activated by depolarizing voltage steps in a cav-1+/+ and cav-1–/– cell. B: paxilline (300 nM) inhibits K+ currents in the same cells illustrated in A. C: paxilline-sensitive K+ currents. D: mean whole cell K+ current density (pA/pF) in cav-1+/+ (n = 11) and cav-1–/– (n = 9) cells. E: paxilline-insensitive K+ current density. F: paxilline-sensitive K+ current density. *P < 0.05 when compared with cav-1+/+ cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

In smooth muscle cells, coupling between voltage-dependent Ca²⁺ channels and RyR channels is "loose" (20). Although structural and molecular mechanisms that establish this coupling process are unclear, loss of voltage-dependent Ca²⁺ channel to RyR channel communication in cav-1^–/– cells likely occurs because of spatial separation of these proteins. Supporting this hypothesis is evidence that although voltage-dependent Ca²⁺ current density was similar in cav-1^+/+ and cav-1^–/– cells, regulation of transient K_Ca currents by voltage-dependent Ca²⁺ channel blockers was abolished in cav-1^–/– cells. In addition, although K_Ca channel density was elevated in cav-1^–/– cells, mean transient K_Ca current amplitude was similar to that in cav-1^+/+ cells. K_Ca channel conductance, Ca²⁺ spark amplitude and spatial spread, the effective coupling of Ca²⁺ sparks to K_Ca channels, and K_Ca channel-apparent Ca²⁺ sensitivity were similar in cav-1^+/+ and cav-1^–/– cells. Caveolae are structural membrane invaginations that may reduce the sarcolemma to SR distance and physically localize RyR channels nearby voltage-dependent Ca²⁺ channels and K_Ca channels in cav-1^+/+ cells. A flattening of the sarcolemma in cav-1^–/– cells may explain the proposed increase in the signaling distance between RyR channels and voltage-dependent Ca²⁺ channels and K_Ca channels. In cav-1^–/– cells, a distant spark would impact a smaller membrane area and induce a lower subsarcolemmal [Ca²⁺]_i elevation. Consistent with our observations, higher K_Ca channel density would be required for distant Ca²⁺ sparks to activate transient K_Ca currents of similar amplitude to those in cav-1^+/+ cells.

Another explanation for the proposed increase in signaling distance is that cav-1 abolishment leads to delocalization of voltage-dependent Ca²⁺ channels and K_Ca channels from within the vicinity of the spark site. Several lines of evidence support such a proposal. In smooth muscle cells, caveolae compartmentalize voltage-dependent Ca²⁺ channels and K_Ca channels (3, 8). In arterial smooth muscle cells, K_Ca channels are proposed not to cluster above Ca²⁺ sparks sites, although in Bufo marinus stomach smooth muscle cells, such clustering may occur (30, 42). In guinea pig bladder smooth muscle, peripheral SR and caveolae are in close proximity, and L-type Ca²⁺ channels located in caveolae strips may be closely opposed to SR RyR channels (25). It is not clear what causes the elevation in K_Ca channel density in cav-1^–/– cells, but in cultured human myometrial smooth muscle cells, K_Ca channels associate with caveolins 1 and 2, and cholesterol depletion with cyclodextrin leads to an increase in whole cell K_Ca current density (3), consistent with the results here. Furthermore, in bovine endothelial cells, cav-1 physically interacts with and inhibits K_Ca channels and this effect can be removed by cholesterol depletion, leading to channel activation (37).

Investigating the effects of chronic caveolae deficiency on smooth muscle Ca²⁺ signaling can provide insights into physiological regulation by these membrane organelles in cav-1^+/+ cells and potential changes that occur during pathophysiology and ontogeny (11, 29, 32, 34, 39). In contrast to the observations here, acute cholesterol depletion with dextrin inhibited arterial smooth muscle Ca²⁺ sparks (23). Acute cholesterol depletion and chronic cav-1 ablation may differentially regulate Ca²⁺ spark frequency through opposing effects on signaling pathways that regulate these events, and in the case of the genetic model, through developmental changes that occur in the sustained absence of cav-1 (10, 39). However, dextrin treatment also induces a change in membrane fluidity that may inhibit Ca²⁺ sparks (26). In agreement, both dextrin and cav-1 ablation abolished coupling between voltage-dependent Ca²⁺ channels and RyR channels that generate sparks (present data and Ref. 23). Whereas dextrin treatment disrupts both caveolar and noncaveolar lipid rafts, in the absence of cav-1, noncaveolar lipid rafts would remain (see Ref. 40). Thus our data suggest that caveolae-localized voltage-dependent Ca²⁺ channels regulate Ca²⁺ sparks in arterial smooth muscle cells. In another previous study, transient K_Ca current frequency was reduced in arterial smooth muscle cells of cav-1^–/– mice (10). It is unclear why data here are contradictory to those observations, but in the previous study, effects of cav-1 deficiency on Ca²⁺ sparks were not measured (10).

Through its amino-terminal scaffolding domain, cav-1 inhibits the activity of many binding partners, including NOS, protein kinase C, and adenylyl cyclase (28, 34). Cav-1 is found in the cytosol (22) and, conceivably, may interact with and inhibit RyR channels. Cav-1 is also found in mitochondria, which regulate Ca²⁺ sparks in cerebral artery smooth muscle cells (7, 41). Cav-1 abolishment may also activate RyR channels indirectly by modulating the activity of signaling pathways that regulate Ca²⁺ sparks (e.g., see Refs. 15 and 41). Data here indicate that cav-1 ablation does not activate Ca²⁺ sparks through NOS activation (31, 33). Because cav-1 ablation may activate RyR channels directly and indirectly, future studies will be required to determine the specific underlying mechanisms. The physiological impact of the transient K_Ca current frequency elevation in cav-1^–/– cells would be a membrane hyperpolarization that reduces voltage-dependent Ca²⁺ channel activity, leading to a decrease in global [Ca²⁺]_i and vasodilation (15). Cav-1^–/– aortas contract less in response to vasoconstrictors and relax more in response to acetylcholine, a vasodilator, when compared with cav-1^+/+ arteries (10, 33), supporting upregulation of a vasodilatory pathway.

In summary, data show that genetic ablation of cav-1 leads to loss of caveolae, an increase in Ca²⁺ spark frequency, abolishment of Ca²⁺ spark regulation by voltage-dependent Ca²⁺ channels, and an increase in K_Ca channel density in cerebral artery smooth muscle cells. In contrast, cav-1 ablation does not attenuate transient K_Ca current activation by Ca²⁺ sparks.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by grants from National Institutes of Health and American Heart Association National Center (to J. H. Jaggar).

	ACKNOWLEDGMENTS

 
We thank Dr. A. Ahmed for technical assistance with electron microscopy and Dr. R. Ostrom for helpful comments on the manuscript. X. Cheng is a recipient of a Predoctoral Fellowship from the Southeast Affiliate of the American Heart Association.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. H. Jaggar, Dept. of Physiology, Univ. of Tennessee Health Science Center, Memphis, TN 38163 (e-mail: jjaggar{at}physio1.utmem.edu)

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. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Babiychuk EB, Smith RD, Burdyga T, Babiychuk VS, Wray S, and Draeger A. Membrane cholesterol regulates smooth muscle phasic contraction. J Membr Biol 198: 95–101, 2004.[ISI][Medline]
2. Berridge MJ, Lipp P, and Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11–21, 2000.[CrossRef][ISI][Medline]
3. Brainard AM, Miller AJ, Martens JR, and England SK. Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K⁺ current. Am J Physiol Cell Physiol 289: C49–C57, 2005.[Abstract/Free Full Text]
4. Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, and Aldrich RW. Vasoregulation by the 1 subunit of the calcium-activated potassium channel. Nature 407: 870–876, 2000.[CrossRef][Medline]
5. Cannell MB, Cheng H, and Lederer WJ. The control of calcium release in heart muscle. Science 268: 1045–1049, 1995.[Abstract/Free Full Text]
6. Cheranov SY and Jaggar JH. Sarcoplasmic reticulum calcium load regulates rat arterial smooth muscle calcium sparks and transient K_Ca currents. J Physiol 544: 71–84, 2002.[Abstract/Free Full Text]
7. Cheranov SY and Jaggar JH. Mitochondrial modulation of Ca²⁺ sparks and transient K_Ca currents in smooth muscle cells of rat cerebral arteries. J Physiol 556: 755–771, 2004.[Abstract/Free Full Text]
8. Darby PJ, Kwan CY, and Daniel EE. Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca²⁺ handling. Am J Physiol Lung Cell Mol Physiol 279: L1226–L1235, 2000.[Abstract/Free Full Text]
9. Doyle DD, Upshaw-Earley J, Bell E, and Palfrey HC. Expression of caveolin-3 in rat aortic vascular smooth muscle cells is determined by developmental state. Biochem Biophys Res Commun 304: 22–25, 2003.[CrossRef][ISI][Medline]
10. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, and Kurzchalia TV. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293: 2449–2452, 2001.[Abstract/Free Full Text]
11. Gratton JP, Bernatchez P, and Sessa WC. Caveolae and caveolins in the cardiovascular system. Circ Res 94: 1408–1417, 2004.[Abstract/Free Full Text]
12. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
13. Isshiki M and Anderson RG. Function of caveolae in Ca²⁺ entry and Ca²⁺-dependent signal transduction. Traffic 4: 717–723, 2003.[CrossRef][ISI][Medline]
14. Jaggar JH. Intravascular pressure regulates local and global Ca²⁺ signaling in cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 281: C439–C448, 2001.[Abstract/Free Full Text]
15. Jaggar JH, Porter VA, Lederer WJ, and Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235–C256, 2000.[Abstract/Free Full Text]
16. Jaggar JH, Stevenson AS, and Nelson MT. Voltage dependence of Ca²⁺ sparks in intact cerebral arteries. Am J Physiol Cell Physiol 274: C1755–C1761, 1998.[Abstract/Free Full Text]
17. Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, Kleppisch T, Rubart M, Stevenson AS, Lederer WJ, Knot HJ, Bonev AD, and Nelson MT. Ca²⁺ channels, ryanodine receptors and Ca²⁺-activated K⁺ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 164: 577–587, 1998.[CrossRef][ISI][Medline]
18. Je HD, Gallant C, Leavis PC, and Morgan KG. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91–H98, 2004.[Abstract/Free Full Text]
19. Knot HJ and Nelson MT. Regulation of arterial diameter and wall [Ca²⁺] in cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol 508: 199–209, 1998.[Abstract/Free Full Text]
20. Kotlikoff MI. Calcium-induced calcium release in smooth muscle: the case for loose coupling. Prog Biophys Mol Biol 83: 171–191, 2003.[CrossRef][ISI][Medline]
21. Li G and Cheung DW. Effects of paxilline on K⁺ channels in rat mesenteric arterial cells. Eur J Pharmacol 372: 103–107, 1999.[CrossRef][ISI][Medline]
22. Li WP, Liu P, Pilcher BK, and Anderson RG. Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria. J Cell Sci 114: 1397–1408, 2001.[Abstract]
23. Lohn M, Furstenau M, Sagach V, Elger M, Schulze W, Luft FC, Haller H, and Gollasch M. Ignition of calcium sparks in arterial and cardiac muscle through caveolae. Circ Res 87: 1034–1039, 2000.[Abstract/Free Full Text]
24. Lopez-Lopez JR, Shacklock PS, Balke CW, and Wier WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science 268: 1042–1045, 1995.[Abstract/Free Full Text]
25. Moore ED, Voigt T, Kobayashi YM, Isenberg G, Fay FS, Gallitelli MF, and Franzini-Armstrong C. Organization of Ca²⁺ release units in excitable smooth muscle of the guinea-pig urinary bladder. Biophys J 87: 1836–1847, 2004.[Abstract/Free Full Text]
26. Munro S. Lipid rafts: elusive or illusive? Cell 115: 377–388, 2003.[CrossRef][ISI][Medline]
27. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633–637, 1995.[Abstract/Free Full Text]
28. Ostrom RS, Liu X, Head BP, Gregorian C, Seasholtz TM, and Insel PA. Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains. Mol Pharmacol 62: 983–992, 2002.[Abstract/Free Full Text]
29. Park DS, Cohen AW, Frank PG, Razani B, Lee H, Williams TM, Chandra M, Shirani J, De Souza AP, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, and Lisanti MP. Caveolin-1 null (–/–) mice show dramatic reductions in life span. Biochemistry 42: 15124–15131, 2003.[CrossRef][Medline]
30. Perez GJ, Bonev AD, and Nelson MT. Micromolar Ca²⁺ from sparks activates Ca²⁺-sensitive K⁺ channels in rat cerebral artery smooth muscle. Am J Physiol Cell Physiol 281: C1769–C1775, 2001.[Abstract/Free Full Text]
31. Porter VA, Bonev AD, Knot HJ, Heppner TJ, Stevenson AS, Kleppisch T, Lederer WJ, and Nelson MT. Frequency modulation of Ca²⁺ sparks is involved in regulation of arterial diameter by cyclic nucleotides. Am J Physiol Cell Physiol 274: C1346–C1355, 1998.[Abstract/Free Full Text]
32. Ratajczak P, Damy T, Heymes C, Oliviero P, Marotte F, Robidel E, Sercombe R, Boczkowski J, Rappaport L, and Samuel JL. Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat. Cardiovasc Res 57: 358–369, 2003.[Abstract/Free Full Text]
33. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russell RG, Li M, Pestell RG, Di VD, Hou H Jr, Kneitz B, Lagaud G, Christ GJ, Edelmann W, and Lisanti MP. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 276: 38121–38138, 2001.[Abstract/Free Full Text]
34. Razani B, Woodman SE, and Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002.[Abstract/Free Full Text]
35. Sanchez M and McManus OB. Paxilline inhibition of the alpha-subunit of the high-conductance calcium-activated potassium channel. Neuropharmacology 35: 963–968, 1996.[CrossRef][ISI][Medline]
36. Wang SQ, Song LS, Lakatta EG, and Cheng H. Ca²⁺ signaling between single L-type Ca²⁺ channels and ryanodine receptors in heart cells. Nature 410: 592–596, 2001.[CrossRef][Medline]
37. Wang XL, Ye D, Peterson TE, Cao S, Shah VH, Katusic ZS, Sieck GC, and Lee HC. Caveolae targeting and regulation of large conductance Ca²⁺-activated K⁺ channels in vascular endothelial cells. J Biol Chem 280: 11656–11664, 2005.[Abstract/Free Full Text]
38. Wellman GC, Santana LF, Bonev AD, and Nelson MT. Role of phospholamban in the modulation of arterial Ca²⁺ sparks and Ca²⁺-activated K⁺ channels by cAMP. Am J Physiol Cell Physiol 281: C1029–C1037, 2001.[Abstract/Free Full Text]
39. Williams TM and Lisanti MP. The caveolin genes: from cell biology to medicine. Ann Med 36: 584–595, 2004.[CrossRef][ISI][Medline]
40. Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M, Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM, Tanowitz HB, and Lisanti MP. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade. J Biol Chem 277: 38988–38997, 2002.[Abstract/Free Full Text]
41. Xi Q, Cheranov SY, and Jaggar JH. Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca²⁺ sparks. Circ Res 97: 354–362, 2005.[Abstract/Free Full Text]
42. Zhuge R, Fogarty KE, Tuft RA, and Walsh JV Jr. Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca²⁺ concentration on the order of 10 µm during a Ca²⁺ spark. J Gen Physiol 120: 15–27, 2002.[Abstract/Free Full Text]

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