CaM-dependent modulation of human Ca1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human Ca1.3 VGCC, however, is largely unexplored.

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel Ca1.2.

The L-type Ca channel Ca1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs Ca1.

Inhibitory effects on L- and N-type calcium channels by a novel Caβ variant identified in a patient with autism spectrum disorder.

Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (Caβ). Our previous findings suggested that activation of L-type VGCCs is a common feature of Caβ subunit mutations found in ASD patients.

Autism-associated mutations in the Caβ calcium-channel subunit increase Ba-currents and lead to differential modulation by the RGK-protein Gem.

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of Caβ subunits of VGCCs.

Single-Channel Resolution of the Interaction between C-Terminal Ca1.3 Isoforms and Calmodulin.

Voltage-dependent calcium (Ca) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of Ca1.

A gating model for wildtype and R1448H Nav1.4 channels in paramyotonia.

We studied the consequences of the Nav1.4 mutation R1448H that is situated in the fourth voltage sensor of the channel and causes paramyotonia, a cold-induced myotonia followed by weakness. Previous work showed that the mutation uncouples inactivation from activation.

Single-channel monitoring of reversible L-type Ca(2+) channel Ca(V)α(1)-Ca(V)β subunit interaction.

Voltage-dependent Ca(2+) channels are heteromultimers of Ca(V)α(1) (pore), Ca(V)β- and Ca(V)α(2)δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca(V)β-isoforms affect gating differentially, and we chose two extremes (Ca(V)β(1a) and Ca(V)β(2b)) regarding single-channel open probability to address this question.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse.

A critical GxxxA motif in the gamma6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current.

The eight members of the calcium channel gamma subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of gamma(2), gamma(3), gamma(4) and gamma(8) (the TARPs) modulate AMPA receptor localization and function, the gamma(1) and gamma(6) subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the gamma(6) subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current.

Gating of the HypoPP-1 mutations: I. Mutant-specific effects and cooperativity.

Hypokalemic periodic paralysis type 1 (HypoPP-1) is a hereditary muscular disorder caused by point mutations in the gene encoding the voltage-gated Ca(2+) channel alpha subunit (Ca(v)1.1). Despite extensive research, the results on HypoPP-1 mutations are minor and controversial, as it is difficult to analyse Ca(2+) channel activation macroscopically due to an existence of two open states.

Gating of the HypoPP-1 mutations: II. Effects of a calcium-channel agonist BayK 8644.

L-type calcium-channel mutations causing hypokalemic periodic paralysis type 1 (HypoPP-1) have pronounced "loss-of-function" features and stabilize the less-selective second open state O(2), as we demonstrated in the companion paper. Here, we compared the effects of the L-type calcium-channel activator (+/-)BayK 8644 (BayK) on the heterologously expressed wild-type (WT) calcium channel, rabbit Cav1.2 HypoPP-1 analogs, and two double mutants (R650H/R1362H, R650H/R1362G).

Affinity of C-reactive protein toward FcgammaRI is strongly enhanced by the gamma-chain.

C-reactive protein (CRP), the prototype human acute phase protein, is widely regarded as a key player in cardiovascular disease, but the identity of its cellular receptor is still under debate. By using ultrasensitive confocal imaging analysis, we have studied CRP binding to transfected COS-7 cells expressing the high-affinity IgG receptor FcgammaRI. Here we show that CRP binds to FcgammaRI on intact cells, with a kd of 10+/-3 micromol/L.

Single-molecule FRET study of denaturant induced unfolding of RNase H.

Single-molecule fluorescence (Förster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population.

Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions.

Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble.