A genetically encoded actuator boosts L-type calcium channel function in diverse physiological settings.

L-type Ca channels (Ca1.2/1.3) convey influx of calcium ions that orchestrate a bevy of biological responses including muscle contraction, neuronal function, and gene transcription.

Decoding polyubiquitin regulation of K7. 1 functional expression with engineered linkage-selective deubiquitinases.

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific.

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain.

Transfer RNA-mediated restoration of potassium current and electrical correction in premature termination long-QT syndrome hERG mutants.

Disease-causing premature termination codons (PTCs) individually disrupt the functional expression of hundreds of genes and represent a pernicious clinical challenge. In the heart, loss-of-function mutations in the hERG potassium channel account for approximately 30% of long-QT syndrome arrhythmia, a lethal cardiac disorder with limited treatment options. Premature termination of ribosomal translation produces a truncated and, for potassium channels, a potentially dominant-negative protein that impairs the functional assembly of the wild-type homotetrameric hERG channel complex.

A Genetically Encoded Actuator Selectively Boosts L-type Calcium Channels in Diverse Physiological Settings.

L-type Ca channels (Ca 1.2/1.3) convey influx of calcium ions (Ca ) that orchestrate a bevy of biological responses including muscle contraction and gene transcription.

Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex.

The slow delayed rectifier potassium current, , conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic up-regulation of requires recruitment of PKA holoenzyme (two regulatory - RI or RII - and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9).

Design and Applications of Genetically-Encoded Voltage-Dependent Calcium Channel Inhibitors.

Ca influx through high-voltage-gated Ca channels (HVGCCs; Ca1/Ca2) is an exceptionally powerful and versatile signal that controls numerous cell and physiological functions including neurotransmission, muscle contraction, and regulation of gene expression. The impressive ability of a singular signal, Ca influx, to have such a plethora of functional outcomes is enabled by: molecular diversity of HVGCC pore-forming α and auxiliary subunits; organization of HVGCCs with extrinsic modulatory and effector protein to form discrete macromolecular complexes with unique properties; distinctive distribution of HVGCCs into separate subcellular compartments; and varying expression profiles of HVGCC isoforms among different tissues and organs. The capacity to block HVGCCs with selectivity and specificity with respect to the different levels of their organization is critical for fully understanding the scope of functional consequences of Ca influx through them, and is also important for realizing their full potential as therapeutic targets.

Perturbation of the host cell Ca homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M.

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO).

Pharmacological rescue of specific long QT variants of KCNQ1/KCNE1 channels.

The congenital Long QT Syndrome (LQTS) is an inherited disorder in which cardiac ventricular repolarization is delayed and predisposes patients to cardiac arrhythmias and sudden cardiac death. LQT1 and LQT5 are LQTS variants caused by mutations in KCNQ1 or KCNE1 genes respectively. KCNQ1 and KCNE1 co-assemble to form critical I potassium channels.

Selective posttranslational inhibition of Caβ-associated voltage-dependent calcium channels with a functionalized nanobody.

Ca influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca influx, to mediate disparate functions is diversity of HVACC pore-forming α and auxiliary Caβ-Caβ subunits. Selective Caα blockers have enabled deciphering their unique physiological roles.

Reduced calcium levels and accumulation of abnormal insulin granules in stem cell models of HNF1A deficiency.

Mutations in HNF1A cause Maturity Onset Diabetes of the Young (HNF1A-MODY). To understand mechanisms of β-cell dysfunction, we generated stem cell-derived pancreatic endocrine cells with hypomorphic mutations in HNF1A. HNF1A-deficient β-cells display impaired basal and glucose stimulated-insulin secretion, reduced intracellular calcium levels in association with a reduction in CACNA1A expression, and accumulation of abnormal insulin granules in association with SYT13 down-regulation.

Targeted ubiquitination of sensory neuron calcium channels reduces the development of neuropathic pain.

Neuropathic pain caused by lesions to somatosensory neurons due to injury or disease is a widespread public health problem that is inadequately managed by small-molecule therapeutics due to incomplete pain relief and devastating side effects. Genetically encoded molecules capable of interrupting nociception have the potential to confer long-lasting analgesia with minimal off-target effects. Here, we utilize a targeted ubiquitination approach to achieve a unique posttranslational functional knockdown of high-voltage-activated calcium channels (HVACCs) that are obligatory for neurotransmission in dorsal root ganglion (DRG) neurons.

Controlling ion channel function with renewable recombinant antibodies.

Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function.

Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg coordination site formed between the C-linker and the S4-S5 linker.

Controlling ion channel trafficking by targeted ubiquitination and deubiquitination.

Plasma membrane-localized ion channels are essential for diverse physiological processes such as neurotransmission, muscle contraction, and osmotic homeostasis. The surface density of such ion channels is a major determinant of their function, and tuning this variable is a powerful way to regulate physiology. Dysregulation of ion channel surface density due to inherited or de novo mutations underlies many serious diseases, and molecules that can correct trafficking deficits are potential therapeutics and useful research tools.

Targeted deubiquitination rescues distinct trafficking-deficient ion channelopathies.

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density, but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF).

The mutation L69P in the PAS domain of the hERG potassium channel results in LQTS by trafficking deficiency.

The congenital long QT syndrome (LQTS) is a cardiac disorder characterized by a prolonged QT interval on the electrocardiogram and an increased susceptibility to ventricular arrhythmias and sudden cardiac death. A frequent cause for LQTS is mutations in the gene (also known as the or ), which reduce or modulate the potassium current I and hence alter cardiac repolarization. In a patient with a clinically diagnosed LQTS, we identified the mutation L69P in the N-terminal PAS (Per-Arnt-Sim) domain of hERG.