Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse.

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.

Two stable cell lines for screening of calcium channel blockers.

Stable cell lines are potentially excellent tools for large-scale screening of new compounds. Two carboxyterminal-deleted constructs of the two splice variants a and b of the calcium channel class C alpha 1 subunit were expressed stably in HEK 293 cells. Each cell line produced regular L-type calcium currents. The opening and closing of the calcium channel elicited by potassium depolarization was followed by Fura-2 transients. These transients were blocked by the calcium channel blocker mibefradil with a concentration for 50% inhibition of 1.7 microM. The cell lines expressing the truncated cardiac alpha 1C-a or smooth muscle alpha 1C-b calcium channel were both blocked by nisoldipine under patch clamp conditions. Nisoldipine interacted with higher affinity with the alpha 1C-b channel than with the alpha 1C-a channel. These results indicate that the two cell lines retain the differential dihydropyridine sensitivity of smooth muscle and cardiac calcium channels and may be potential tools for the screening of L-type calcium channel blockers.

Subunit-dependent modulation of recombinant L-type calcium channels. Molecular basis for dihydropyridine tissue selectivity.

At least four calcium channel subtypes (P, T, N, and L) have now been classified on the basis of their biophysical and/or pharmacological properties. L-type channels, a channel family particularly important to physiological function of the cardiovascular system, are identified by their slow voltage- and calcium-dependent inactivation as well as their sensitivity to dihydropyridine (DHP) calcium channel antagonists. In this study, we report the results of experiments in which we have measured the DHP modulation of recombinant calcium channel activity in cells transfected with alpha 1 subunits of cardiac and smooth muscle L-type calcium channels. We find subunit-dependent differences in the voltage and concentration dependence of channel modulation. Our results provide evidence for a molecular basis for DHP sensitivity of heart and smooth muscle calcium channels and, additionally, indicate that, even within one family of calcium channels, slight differences in channel structure can cause marked differences in channel pharmacology.

Stable co-expression of calcium channel alpha 1, beta and alpha 2/delta subunits in a somatic cell line.

1. The high-voltage-activated L-type calcium channel is a multi-protein complex of alpha 1, alpha 2/delta, beta and gamma subunits. The alpha 1 subunit contains the voltage-dependent calcium-conducting pore. Chinese hamster ovary (CHO) cells were stably transfected with the complementary DNA of the alpha 1, beta and alpha 2/delta subunits. These subunits were not detected in wild-type CHO cells. 2. The alpha 1 (CaCh2b) subunit itself directed the expression of functional calcium channels which bound calcium channel blockers and showed voltage-dependent activation and inactivation. 3. The co-expression of the alpha 1 subunit with the beta subunit (CaB1 gene) enhanced the density of the dihydropyridine binding sites 2- to 3-fold and increased dihydropyridine-sensitive barium inward currents (IBa) up to 3.5-fold from -13.3 microA/cm2 (alpha 1 subunit) to -46.7 microA/cm2 (alpha 1 and beta subunits). 4. Co-expression of the beta subunit did not change the sensitivity of IBa towards dihydropyridines, but accelerated current activation and inactivation and shifted the half-maximal steady-state activation and inactivation to slightly more hyperpolarizing potentials. 5. The co-expression of the alpha 2/delta subunit together with alpha 1 and beta subunits accelerated the inactivation kinetics of the channel without a major effect on the other parameters. 6. These results indicate that the beta and alpha 2/delta subunit interact with the alpha 1 subunit and modulate thereby the properties of the alpha 1 subunit-dependent inward current.

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.