Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Influence of channel subunit composition on L-type Ca2+ current kinetics and cardiac wave stability

Previous studies have demonstrated that the slope of the function relating the action potential duration (APD) and the diastolic interval, known as the APD restitution curve, plays an important role in the initiation and maintenance of ventricular fibrillation. Since the APD restitution slope critically depends on the kinetics of the L-type Ca(2+) current, we hypothesized that manipulation of the subunit composition of these channels may represent a powerful strategy to control cardiac arrhythmias. We studied the kinetic properties of the human L-type Ca(2+) channel (Ca(v)1.2) coexpressed with the alpha(2)delta-subunit alone (alpha(1C) + alpha(2)delta) or in combination with beta(2a), beta(2b), or beta(3) subunits (alpha(1C) + alpha(2)delta + beta), using Ca(2+) as the charge carrier. We then incorporated the kinetic properties observed experimentally into the L-type Ca(2+) current mathematical model of the cardiac action potential to demonstrate that the APD restitution slope can be selectively controlled by altering the subunit composition of the Ca(2+) channel. Assuming that beta(2b) most closely resembles the native cardiac L-type Ca(2+) current, the absence of beta, as well as the coexpression of beta(2a), was found to flatten restitution slope and stabilize spiral waves. These results imply that subunit modification of L-type Ca(2+) channels can potentially be used as an antifibrillatory strategy.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Expression of Ca(2+) channel subunits during cardiac ontogeny in mice and rats: identification of fetal alpha(1C) and beta subunit isoforms

Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.     

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.     

Regulation of Ca-sensitive inactivation of a 1-type Ca2+ channel by specific domains of beta subunits.

Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.     

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.