José M. Fernández-Fernández

Gain-of-function mutation in the KCNMB1 potassium channel subunit is associated with low prevalence of diastolic hypertension

Hypertension is the most prevalent risk factor for cardiovascular diseases, present in almost 30% of adults. A key element in the control of vascular tone is the large-conductance, Ca(2+)-dependent K(+) (BK) channel. The BK channel in vascular smooth muscle is formed by an ion-conducting alpha subunit and a regulatory beta(1) subunit, which couples local increases in intracellular Ca(2+) to augmented channel activity and vascular relaxation. Our large population-based genetic epidemiological study has identified a new single-nucleotide substitution (G352A) in the beta(1) gene (KCNMB1), corresponding to an E65K mutation in the protein. This mutation results in a gain of function of the channel and is associated with low prevalence of moderate and severe diastolic hypertension. BK-beta(1E65K) channels showed increased Ca(2+) sensitivity, compared with wild-type channels, without changes in channel kinetics. In conclusion, the BK-beta(1E65K) channel might offer a more efficient negative-feedback effect on vascular smooth muscle contractility, consistent with a protective effect of the K allele against the severity of diastolic hypertension.

TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity

Autoregulation of the ciliary beat frequency (CBF) has been proposed as the mechanism used by epithelial ciliated cells to maintain the CBF and prevent the collapse of mucociliary transport under conditions of varying mucus viscosity. Despite the relevance of this regulatory response to the pathophysiology of airways and reproductive tract, the underlying cellular and molecular aspects remain unknown. Hamster oviductal ciliated cells express the transient receptor potential vanilloid 4 (TRPV4) channel, which is activated by increased viscous load involving a phospholipase A2–dependent pathway. TRPV4-transfected HeLa cells also increased their cationic currents in response to high viscous load. This mechanical activation is prevented in native ciliated cells loaded with a TRPV4 antibody. Application of the TRPV4 synthetic ligand 4α-phorbol 12,13-didecanoate increased cationic currents, intracellular Ca2+, and the CBF in the absence of a viscous load. Therefore, TRPV4 emerges as a candidate to participate in the coupling of fluid viscosity changes to the generation of the Ca2+ signal required for the autoregulation of CBF.

Constitutive Activation of G-proteins by Polycystin-1 Is Antagonized by Polycystin-2

Polycystin-1 (PC1), a 4,303-amino acid integral membrane protein of unknown function, interacts with polycystin-2 (PC2), a 968-amino acid α-type channel subunit. Mutations in their respective genes cause autosomal dominant polycystic kidney disease. Using a novel heterologous expression system and Ca2+ and K+ channels as functional biosensors, we found that full-length PC1 functioned as a constitutive activator of Gi/o-type but not Gq-type G-proteins and modulated the activity of Ca2+ and K+ channels via the release of Gβγ subunits. PC1 lacking the N-terminal 1811 residues replicated the effects of full-length PC1. These effects were independent of regulators of G-protein signaling proteins and were lost in PC1 mutants lacking a putative G-protein binding site. Co-expression with full-length PC2, but not a C-terminal truncation mutant, abrogated the effects of PC1. Our data provide the first experimental evidence that full-length PC1 acts as an untraditional G-protein-coupled receptor, activity of which is physically regulated by PC2. Thus, our study strongly suggests that mutations in PC1 or PC2 that distort the polycystin complex would initiate abnormal G-protein signaling in autosomal dominant polycystic kidney disease.

Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking.

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (Δ384–444) variants and the new TRPV4-C (Δ237–284), TRPV4-D (Δ27–61), and TRPV4-E (Δ237–284 and Δ384–444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5′-6′-epoxyeicosatrienoic acid

Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2–EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)–inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.

Protective effect of the KCNMB1 E65K genetic polymorphism against diastolic hypertension in aging women and its relevance to cardiovascular risk.

The E65K polymorphism in the &bgr;1-subunit of the large-conductance, Ca2+-dependent K+ (BK) channel, a key element in the control of arterial tone, has recently been associated with low prevalence of diastolic hypertension. We now report the modulatory effect of sex and age on the association of the E65K polymorphism with low prevalence of diastolic hypertension and the protective role of E65K polymorphism against cardiovascular disease. We analyzed the genotype frequency of the E65K polymorphism in 3924 participants selected randomly in two cross-sectional studies. A five-year follow-up of the cohort was performed to determine whether cardiovascular events had occurred since inclusion. Estrogen modulation of wild-type and mutant ion channel activity was assessed after heterologous expression and electrophysiological studies. Multivariate regression analyses showed that increasing age upmodulates the protective effect of the K allele against moderate-to-severe diastolic hypertension in the overall group of participants (odds ratio [OR], 0.35; P=0.006). The results remained significant when analyses were restricted to women (OR, 0.18; P=0.02) but not men (OR, 0.46; P=0.09). This effect was independent of the reported acute modulation of BK channels by estrogen. A five-year follow-up study also demonstrated a reduced age- and sex-adjusted hazard ratio of 0.11, 95% CI, 0.01 to 0.79 of K-carriers for “combined cardiovascular disease” (myocardial infarction and stroke) compared with EE homozygotes. Our study provides the first genetic evidence for the different impact of the BK channel in the control of human blood pressure in men and women, with particular relevance in aging women, and highlights the E65K polymorphism as one of the strongest genetic factors associated thus far to protection against myocardial infarction and stroke.

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis.

Background: T-type calcium channels play a unique role in low-threshold exocytosis. Results: Syntaxin-1A interacts with the carboxyl terminus domain of Cav3.2 channels and modulates channel activity and low-threshold exocytosis. Conclusion: Low-threshold exocytosis relies on a syntaxin-1A/T-type calcium channel signaling complex. Significance: Elucidating the molecular mechanisms by which T-type channels control low-threshold exocytosis is crucial for understanding their implication in synaptic transmission. T-type calcium channels represent a key pathway for Ca2+ entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Cav3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Cav3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Cav3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca2+ channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

A loss-of-function nonsynonymous polymorphism in the osmoregulatory TRPV4 gene is associated with human hyponatremia

Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential vanilloid 4 (TRPV4) channel is postulated to comprise an element of the central tonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. We show that this polymorphism is significantly associated with serum sodium concentration and with hyponatremia (serum sodium concentration ≤135 mEq/L) in 2 non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration is lower among subjects with the TRPV4P19S allele relative to the wild-type allele. Subjects with the minor allele were 2.4−6.4 times as likely to exhibit hyponatremia as subjects without the minor allele (after inclusion of key covariates). Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4P19S polymorphism showed diminished response to hypotonic stress (relative to the wild-type channel) and to the osmotransducing lipid epoxyeicosatrienoic acid in heterologous expression studies. These data suggest that this polymorphism affects TRPV4 function in vivo and likely influences systemic water balance on a population-wide basis.

Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines.

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BKCa) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch–clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca2+ entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4α-phorbol 12,13-didecanoate (4αPDD). Cell-attached and whole-cell patch–clamp of HBE cells exposed to 4αPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BKCa channels subsequent to extracellular Ca2+ influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BKCa modulation after TRPV4 activation showed that the Ca2+ signal can be generated away from the BKCa location at the plasma membrane, and it is not mediated by intracellular Ca2+ release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BKCa in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BKCa in response to high-viscous solutions.

Plasma membrane voltage-dependent anion channel mediates antiestrogen-activated maxi Cl- currents in C1300 neuroblastoma cells.

The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl– channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl– channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl– current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin β subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl– currents, suggesting that VDAC forms the plasma membrane Maxi Cl– channel or a part thereof.