Abstract
Skeletal muscle voltage-gated Na
+
channel (Na
V
1.4) activity is subject to calmodulin (CaM) mediated Ca
2+
-dependent inactivation; no such inactivation is observed in the cardiac Na
+
...channel (Na
V
1.5). Taken together, the crystal structures of the Na
V
1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca
2+
-dependent CaM N-lobe binding site previously identified in Na
V
1.5 is not present in Na
V
1.4 allowing the N-lobe to signal other regions of the Na
V
1.4 channel. Consistent with this mechanism, removing this binding site in Na
V
1.5 unveils robust Ca
2+
-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca
2+
-dependent inactivation is effected by CaM’s N-lobe binding outside the Na
V
C-terminal while CaM’s C-lobe remains bound to the Na
V
C-terminal. As the N-lobe binding motif of Na
V
1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.
Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and ...proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221-O-GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation.
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•TRPC6 is constitutively O-GlcNAcylated at T221 to suppress basal conductance•A T221A mutant TRPC6 has much greater conductance and NFAT-activation•Reducing but not raising O-GlcNAc alters T221-dependent TRPC6 function•T221 coordinates with S199, E200, and E246 to control basal channel activity
Biochemistry; Cellular physiology; Cell biology
The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A, and regulatory proteins that reduce or ...eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H.
In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak INa and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac INa; WT and mutant INa decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels.
We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.
Abstract only O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling ...proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.
Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum ...disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking.
To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing.
Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons.
Taken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.
Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes ...and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRSs) for schizophrenia (SCZ) and a clinical diagnosis of SCZ, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures in the patient-derived neurons that implicated altered Na
channel function, action potential interspike interval, and gamma-aminobutyric acid-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these SCZ patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.
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