Direct modulation of TRPC ion channels by Gα proteins.

GPCR-Gi protein pathways are involved in the regulation of vagus muscarinic pathway under physiological conditions and are closely associated with the regulation of internal visceral organs. The muscarinic receptor-operated cationic channel is important in GPCR-Gi protein signal transduction as it decreases heart rate and increases GI rhythm frequency. In the SA node of the heart, acetylcholine binds to the M2 receptor and the released Gßγ activates GIRK (I(K,ACh)) channel, inducing a negative chronotropic action. In gastric smooth muscle, there are two muscarinic acetylcholine receptor (mAChR) subtypes, M2 and M3. M2 receptor activates the muscarinic receptor-operated nonselective cationic current (mIcat, NSCC(ACh)) and induces positive chronotropic effect. Meanwhile, M3 receptor induces hydrolysis of PIP2 and releases DAG and IP3. This IP3 increases intracellular Ca2+ and then leads to contraction of GI smooth muscles. The activation of mIcat is inhibited by anti-Gi/o protein antibodies in GI smooth muscle, indicating the involvement of Gαi/o protein in the activation of mIcat. TRPC4 channel is a molecular candidate for mIcat and can be directly activated by constitutively active Gαi QL proteins. TRPC4 and TRPC5 belong to the same subfamily and both are activated by Gi/o proteins. Initial studies suggested that the binding sites for G protein exist at the rib helix or the CIRB domain of TRPC4/5 channels. However, recent cryo-EM structure showed that IYY58-60 amino acids at ARD of TRPC5 binds with Gi3 protein. Considering the expression of TRPC4/5 in the brain, the direct G protein activation on TRPC4/5 is important in terms of neurophysiology. TRPC4/5 channels are also suggested as a coincidence detector for Gi and Gq pathway as Gq pathway increases intracellular Ca2+ and the increased Ca2+ facilitates the activation of TRPC4/5 channels. More complicated situation would occur when GIRK, KCNQ2/3 (IM) and TRPC4/5 channels are co-activated by stimulation of muscarinic receptors at the acetylcholine-releasing nerve terminals. This review highlights the effects of GPCR-Gi protein pathway, including dopamine, µ-opioid, serotonin, glutamate, GABA, on various oragns, and it emphasizes the importance of considering TRPC4/5 channels as crucial players in the field of neuroscience.

ADPKD-Causing Missense Variants in Polycystin-1 Disrupt Cell Surface Localization or Polycystin Channel Function.

Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of this monogenic disorder, our limited mechanistic understanding of ADPKD has hindered therapeutic development. Here, we successfully developed bioassays that functionally classify missense variants in polycystin-1 (PC1). Strikingly, ADPKD pathogenic missense variants cluster into two major categories: 1) those that disrupt polycystin cell surface localization or 2) those that attenuate polycystin ion channel activity. We found that polycystin channels with defective surface localization could be rescued with a small molecule. We propose that small-molecule-based strategies to improve polycystin cell surface localization and channel function will be effective therapies for ADPKD patients.

Pore residues of transient receptor potential channels canonical 1 and 4 heteromer determine channel properties.

Transient receptor potential channels canonical 1 and 4 (TRPC1 and TRPC4) are proteins belonging to the same TRPC channel family, and the two are known to form a heterotetrameric channel. TRPC4 can form a homotetrameric, nonselective cation channel by itself, but the involvement of the TRPC1 subunit changes several major characteristics of the channel. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4 as a determinant of the identity and characteristics of a heteromeric TRPC1/4 channel: decreased calcium permeability of the channel and outward-rectifying current-voltage (I-V) curve. Mutants and chimeras of the pore residues were created, and their currents were recorded using whole cell patch clamp. The lower gate mutants of TRPC4 exhibited diminished calcium permeability as measured by GCaMP6 fluorescence. Also, chimeric channels substituting the pore region of TRPC1 to TRPC4 were made to locate the pore region that is critical in the production of an outward-rectifying I-V curve characteristic of TRPC1/4 heteromeric channels.NEW & NOTEWORTHY Heteromer research has been a challenging field due to lack of structural studies. Using chimeras and single mutants, we present evidence that the pore region of TRPC1/4 heteromer contributes to determining the channel's characteristics such as calcium permeability, I-V curve, and conductance.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.