Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1) 4 (KCNE1) n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or I Ks , is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether I Ks is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments I Ks current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1) 4 (KCNE1) n channels are present as components of I Ks and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.cardiac physiology | drug | long QT | pharmacology
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery.
T-type Ca2+ channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca2+ channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca2+ channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620) a selective T-Type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-Type calcium current, inhibition of low threshold spike and rebound burst activity. Based on the basal ganglia circuitry in Parkinson’s disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca2+ channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A2A antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca2+ function in vitro and in vivo, and freely available.
TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative porelining basic residues significantly altered the IC 50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.calcium-activated channels | chloride channels | channel pharmacology | TMEM16A | ion channel biophysics
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