L-type calcium channel α-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current

Cardiac voltage-gated L-type Ca channels (CaV) are multiprotein complexes, including accessory subunits such as CaVβ2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel α-subunit (CaV1.2) to CaVβ2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies CaVβ2-Rem inhibition of Ca channel current. We first tested the effect of Rem on CaV1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with CaV1.2 reduces Ba current expression under basal conditions, and CaVβ2a coexpression enhances Rem block of CaV1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 μM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without CaVβ2a. To test whether the H-89 action was a consequence of the phosphorylation status of CaV1.2, we examined Rem regulation of the PKA-insensitive CaV1.2 serine 1928 (S1928) to alanine mutation (CaV1.2-S1928A). CaV1.2-S1928A current was not inhibited by Rem and when coexpression with CaVβ2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the CaV1.2 COOH terminus contributes to Rem-dependent channel inhibition.

Carbenoxolone Inhibition of Voltage-Gated Ca Channels and Synaptic Transmission in the Retina

We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 μM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC50 of 48 μM. At 100 μM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca2+] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Müller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback.

Calcium Channel Activation Facilitated by Nitric Oxide in Retinal Ganglion Cells

We investigated the modulation of voltage-gated Ca channels by nitric oxide (NO) in isolated salamander retinal ganglion cells with the goals of determining the type of Ca channel affected and the signaling pathway by which modulation might occur. The NO donors, S-nitroso- N-acetyl-penicillamine (SNAP, 1 mM) and S-nitroso-cysteine (1 mM) induced modest increases in the amplitude of Ca channel currents recorded with ruptured- and permeabilized-patch techniques by causing a subpopulation of the Ca channels to activate at more negative potentials. The Ca channel antagonists ω-conotoxin GVIA and nisoldipine each reduced the Ca channel current partially, but only ω-conotoxin GVIA blocked the enhancement by SNAP. The SNAP-induced increase was blocked by oxadiazolo-quinoxaline (50 μM), suggesting that the NO generated by SNAP acts via a soluble guanylyl cyclase to raise levels of cGMP. The membrane-permeant cGMP analog 8-(4-chlorophenylthio) guanosine cyclic monophosphate also enhanced Ca channel currents and 8-bromo guanosine cyclic monophosphate (1 mM) occluded enhancement by SNAP. Consistent with these results, isobutyl-methyl-xanthine (IBMX, 10 μM), which can raise cGMP levels by inhibiting phosphodiesterase activity, increased Ca channel current by the same amount as SNAP and occluded subsequent enhancement by SNAP. Neither IBMX, the cGMP analogs, nor SNAP itself, led to activation of cGMP-gated channels. N-[2-(methylamino)ethyl]−5-isoquinoline-sulfonamide (2 μM), a broad spectrum inhibitor of protein kinase activity, KT5823 (1 μM), a specific protein kinase G (PKG) inhibitor, and a peptide inhibitor of PKG (200 μM) blocked SNAP enhancement, as did 5′-adenylylimidophosphate (1.5 mM), a nonhydrolyzable ATP analog that prevents protein phosphorylation. A peptide inhibitor of protein kinase A (10 nM) did not block the facilitory effects of SNAP. Okadaic acid (1 μM), a phosphatase inhibitor, had no effect by itself but increased the enhancement of Ca channel current by SNAP. These results suggest that NO modulates retinal ganglion cell N-type Ca channels by facilitating their voltage-dependent activation via a mechanism involving guanylyl cyclase/PKG-dependent phosphorylation. This effect could fine-tune neural integration in ganglion cells or play a role in ganglion cell disease by modulating intracellular calcium signaling.

Inhibition by external Cd2+ of Na/Ca exchange and L-type Ca channel in rabbit ventricular myocytes

We investigated the effect of external Cd2+ on the Na/Ca exchange and the L-type Ca channel current (ICa,L) in whole cell patch-clamped rabbit ventricular myocytes at 36 degrees C. After the interfering ion channels and the Na/K pump were blocked, the exchange current was measured as the membrane current that was inhibited by 5 mM nickel. External Cd2+ inhibited Na/Ca exchange with a dissociation constant (KD) of 320.6 +/- 12.4 microM and a Hill coefficient of 1.5 +/- 0.09 (n = 13 cells) and ICa,L with a KD of 2.14 +/- 0.15 microM and a Hill coefficient of 0.74 +/- 0.03 (n = 11 cells). We observed some overlap in the Cd2+ concentration that blocked each mechanism. Cd2+ (100-500 microM) is used commonly to block ICa,L completely. However, 100 microM Cd2+ also inhibits 20% of the Na/Ca exchange activity, whereas 500 microM Cd2+ inhibits 60%.

The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors.

High-voltage activated Ca channels in tiger salamander cone photoreceptors were studied with nystatin-permeabilized patch recordings in 3 mM Ca2+ and 10 mM Ba2+. The majority of Ca channel current was dihydropyridine sensitive, suggesting a preponderance of L-type Ca channels. However, voltage-dependent, incomplete block (maximum 60%) by nifedipine (0.1-100 microM) was evident in recordings of cones in tissue slice. In isolated cones, where the block was more potent, nifedipine (0.1-10 microM) or nisoldipine (0.5-5 microM) still failed to eliminate completely the Ca channel current. Nisoldipine was equally effective in blocking Ca channel current elicited in the presence of 10 mM Ba2+ (76% block) or 3 mM Ca2+ (88% block). 15% of the Ba2+ current was reversibly blocked by omega-conotoxin GVIA (1 microM). After enhancement with 1 microM Bay K 8644, omega-conotoxin GVIA blocked a greater proportion (22%) of Ba2+ current than in control. After achieving partial block of the Ba2+ current with nifedipine, concomitant application of omega-conotoxin GVIA produced no further block. The P-type Ca channel blocker, omega-agatoxin IVA (200 nM), had variable and insignificant effects. The current persisting in the presence of these blockers could be eliminated with Cd2+ (100 microM). These results indicate that photoreceptors express an L-type Ca channel having a distinguishing pharmacological profile similar to the alpha 1D Ca channel subtype. The presence of additional Ca channel subtypes, resistant to the widely used L-, N-, and P-type Ca channel blockers, cannot, however, be ruled out.

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.