SM22␣ is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22␣, gene targeting was used to generate SM22␣-deficient (SM22 ؊/؊LacZ ) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22␣ initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22␣ transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22 ؉/؊LacZ embryos revealed detectable ␤-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, ␤-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22␣-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22␣؉/؊ and SM22␣ ؉/؉ littermates. The vasculature and SMC-containing tissues of SM22␣-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22␣ is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.

Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the alpha1B, alpha2a, and beta1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than -30 mV, and were strongly blocked by the N-type channel blocker omega-conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0. 72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than -50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ >/= Cd2+ >> Zn2+ > Ni2+ >/= Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 x 10(8) M-1 s-1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22␣ promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22␣ promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5 of the minimal SM22␣ promoter (base pairs ؊90 to ؉41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22␣ promoter inactivates the 441-base pair SM22␣ promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

Voltage-dependent calcium channels are located in the plasma membrane and form a highly selective conduit by which Ca2+ ions enter all excitable cells and some nonexcitable cells. Extensive characterization studies have revealed the existence of one low (T) and five high-voltage-activated calcium channel types (L, N, P, Q, and R). The high voltage-activated calcium channels have been found to exist as heteromultimers, consisting of an alpha1, beta, alpha2/delta, and gamma subunit. Molecular cloning has revealed the existence of 10 channel transcripts, and expression of these cloned calcium channel genes has shown that basic voltage-activated calcium channel function is strictly carried by the corresponding alpha1 subunits. In turn, the auxiliary subunits serve to modulate calcium channel function by altering the voltage dependence of channel gating, kinetics, and current amplitude, thereby creating a likelihood for calcium channels with multiple properties. Although for calcium channels to be effective, Ca2+ ions must enter selectively through the pore of the alpha1-subunit, bypassing competition with other extracellular ions. The structural determinants of this highly selective Ca2+ filter reside within the four glutamic acid residues located at homologous positions within each of the four pore-forming segments. Together, these residues form a single or multiple Ca2+ affinity site(s) that entrap calcium ions, which are then electrostatically repulsed through the intracellular opening of the pore. This mechanism of high-selectivity calcium filtration, the spatial arrangement of pore glutamic acid residues, and the coordination chemistry of calcium binding are discussed in this review.