Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels

Cav1.4 channels are unique among the high voltage-activated Ca²⁺ channel family because they completely lack Ca²⁺-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca²⁺ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca²⁺-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca²⁺-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca²⁺ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca²⁺ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca²⁺-dependent inactivation, whereas it does not interfere with other calmodulin effects.Retinal photoreceptors and bipolar cells contain a highly specialized type of synapse designated ribbon synapses. Glutamate release in these synapses is controlled via graded and sustained changes in membrane potential that are maintained throughout the duration of a light stimulus (1, 2). In recent years, it became clear that Cav1.4 L-type Ca²⁺ channels are the main channel subtype converting these analog input signals into corresponding permanent glutamate release (1, 3–5). In support of this mechanism, mutations in the Cav1.4 gene have been identified in patients suffering from congenital stationary night blindness type 2 and X-linked cone rod dystrophy (6–8). Individuals displaying congenital stationary night blindness type 2 as well as mice deficient in Cav1.4 typically have abnormal electroretinograms that indicate a loss of neurotransmission from the rods to second order bipolar cells, which is attributable to a loss of Cav1.4 (3).Retinal Cav1.4 channels are set apart from other high voltage-activated (HVA)³ Ca²⁺ channels by their total lack of Ca²⁺-dependent inactivation (CDI) and their very slow voltage-dependent inactivation (VDI). Recently, we and others discovered an inhibitory domain (ICDI: inhibitor of CDI) in the C-terminal tail of the Cav1.4 channel that eliminates Ca²⁺-dependent inactivation in this channel by binding to upstream regulatory motifs (9, 10). Importantly, introducing the ICDI into the backbone of Cav1.2 or Cav1.3 almost completely abolishes the CDI of these channels. Contrasting with the clear cut function, the underlying mechanism by which ICDI abolishes CDI remains controversial. It was suggested that ICDI displaces the Ca²⁺ sensor calmodulin (CaM) from binding to the proximal C terminus (10), suggesting that the binding sites of CaM and ICDI are largely overlapping or allosterically coupled to each other. Alternatively, our own data rather suggested that CaM and the ICDI domain bind to different portions of the proximal C terminus (9). We proposed that the interaction between the ICDI domain and the EF-hand, a motif with a central role for transducing CDI (11–16), switches off CDI without impairing binding of CaM to the channel. In this study, we designed experiments to differentiate between these two models. Here, using FRET in HEK293 cells, we provide evidence that in living cells, CaM is bound to the full-length C terminus of Cav1.4 (i.e. in the presence of ICDI). Furthermore, our data suggest that the steric orientation of the CaM/Cav channel complex differs between Cav1.2 and Cav1.4 channels. We show that CaM preassociation with Cav1.4 controls current density and also affects VDI. Thus, although CaM does not trigger CDI in Cav1.4 as it does in other HVA Ca²⁺ channels, it is still an important regulator of this channel.     

Function and Dysfunction of CNG Channels: Insights from Channelopathies and Mouse Models

Channels directly gated by cyclic nucleotides (CNG channels) are important cellular switches that mediate influx of Na⁺ and Ca²⁺ in response to increases in the intracellular concentration of cAMP and cGMP. In photoreceptors and olfactory receptor neurons, these channels serve as final targets for cGMP and cAMP signaling pathways that are initiated by the absorption of photons and the binding of odorants, respectively. CNG channels have been also found in other types of neurons and in non-excitable cells. However, in most of these cells, the physiological role of CNG channels has yet to be determined. CNG channels have a complex heteromeric structure. The properties of individual subunits that assemble in specific stoichiometries to the native channels have been extensively investigated in heterologous expression systems. Recently, mutations in human CNG channel genes leading to inherited diseases (so-called channelopathies) have been functionally characterized. Moreover, mouse knockout models were generated to define the role of CNG channel proteins in vivo. In this review, we will summarize recent insights into the physiological and pathophysiological role of CNG channel proteins that have emerged from genetic studies in mice and humans.     

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.     

Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.