Interspecies differences in PTH-mediated PKA phosphorylation of the epithelial calcium channel TRPV5

The epithelial calcium (Ca2+) channel TRPV5 (transient receptor potential vanilloid 5) is expressed in the distal convoluted tubule of the kidney and facilitates active Ca2+ reabsorption. This process is instrumental for the maintenance of Ca2+ homeostasis. Therefore, all aspects of TRPV5 function are tightly regulated by the calciotropic parathyroid hormone (PTH). Rabbit (rb)TRPV5 channel activity was shown to be stimulated upon PTH-mediated protein kinase A (PKA) phosphorylation. Since there is incomplete conservation of the PKA consensus motif (RR/QxT) across species, the aim of this study was to extend these findings to humans and characterize the expression and function of human (h)TRPV5. Functional differences between rbTRPV5 and hTRPV5 upon PTH stimulation were investigated using 45Ca2+ uptake assays, Fura-2 Ca2+ imaging, and cell surface biotinylation. While PTH treatment enhanced rbTRPV5 channel activity, it did not stimulate hTRPV5 activity. Mutation of the human RQxT motif into rabbit RRxT (hTRPV5 Q706R) partially restored the sensitivity to PTH. An ancestral sequence reconstruction of TRPV5 orthologues demonstrated that the change in the RRxT motif coincides with the creation of another putative PKA motif (RGAS to RRAS) in the amino terminus of hTRPV5. Interestingly, a constitutively phosphorylated hTRPV5 mutant (hTRPV5 S141D) displayed significantly decreased channel function, while its plasma membrane abundance was increased. Taken together, PTH-mediated stimulation of TRPV5, via PKA, is not conserved in humans. Our data suggest that PTH regulation of TRPV5 is altered in humans, an important observation for future studies that may add to new concepts on the role of PTH in renal Ca2+ handling

Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway

A de novo KCNA1 Mutation in a Patient with Tetany and Hypomagnesemia

Mutations in the KCNA1 gene encoding the voltage-gated potassium (K+) channel Kv1.1 have been linked to rare neurological syndromes, episodic ataxia type 1 (EA1) and myokymia. In 2009, a KCNA1 mutation was identified in a large family with autosomal dominant hypomagnesemia. Despite efforts in establishing a genotype-phenotype correlation for the wide variety of symptoms in EA1, little is known on the serum magnesium (Mg2+) levels in these patients. In the present study, we describe a new de novo KCNA1 mutation in a Polish patient with tetany and hypomagnesemia. Electrophysiological and biochemical analyses were performed to determine the pathogenicity of the mutation. A female patient presented with low serum Mg2+ levels, renal Mg2+ wasting, muscle cramps, and tetanic episodes. Whole exome sequencing identified a p. Leu328Val mutation in KCNA1 encoding the Kv1.1 K+ channel. Electrophysiological examinations demonstrated that the p. Leu328Val mutation caused a dominant-negative loss of function of the encoded Kv1.1 channel. Cell surface biotinylation showed normal plasma membrane expression. Taken together, this is the second report linking KCNA1 with hypomagnesemia, thereby emphasizing the need for further evaluation of the clinical phenotypes observed in patients carrying KCNA1 mutations. (C) 2018 S. Karger AG, Base

Proposed model of flavaglines action.

<p>Flavaglines stimulate TRPM6 activity by acting on downstream effector(s) of the insulin receptor. PHB2 and CDK5, proteins which are known to regulate TRPM6 are localized in lipid rafts.</p

Flavaglines act upon a common pathway with insulin receptor signaling.

<p><b>a-d.</b> The average time-course of current development of cells pre-incubated with (full symbols) or without (empty symbols) FL23 (50 nM) for: (<b>a</b>) TRPM6^V1393I (n≥8), (<b>b</b>) TRPM6^K1584E (n≥7), (<b>c</b>) wild type TRPM6 together with Rac1^T17N (n≥6) and (<b>d</b>) wild type TRPM6 together with Rac1^G12V (n≥11). <b>e.</b> FL23 pre-incubation failed to alter currents in cells expressing TRPM6^V1393I (n = 11), TRPM6^K1584E (n≥8) and cells co-expressing wild type TRPM6 together with Rac1^T17N (n = 8) and TRPM6 together with Rac1^G12V (n≥14). Right panel shows current values normalized to each control condition. Stars indicate statistically significant difference (P<0.05) between vehicle- (white bar) and compound-treated cells (black bar).</p

Analogs of FL23 show distinct effects on TRPM6 currents.

<p><b>a.</b> Typical current-voltage curves obtained 200 s after break-in are shown for vehicle and FL23 (50 nM) pre-incubated cells. <b>b.</b> The average time-course of TRPM6 current development with (n = 8) or without (n = 11) FL23 (50 nM) is shown for current values measured at +80 mV. <b>c.</b> The average time-course of TRPM6 current development with FL2 (50 nM, n = 12), vehicle (n = 10) or FL23 (50 nM, n = 10) pre-treatment is shown for current values measured at +80 mV. <b>d.</b> FL2 incubation did not significantly stimulate TRPM6 activity (n≥10). Stars indicate statistically significant difference (P<0.05) between vehicle and compound-treated cells.</p