Shorelines at the interface of marine, estuarine and terrestrial biomes are among the most degraded and threatened habitats in the coastal zone because of their sensitivity to sea level rise, storms ...and increased human utilization. Previous efforts to protect shorelines have largely involved constructing bulkheads and seawalls which can detrimentally affect nearshore habitats. Recently, efforts have shifted towards "living shoreline" approaches that include biogenic breakwater reefs. Our study experimentally tested the efficacy of breakwater reefs constructed of oyster shell for protecting eroding coastal shorelines and their effect on nearshore fish and shellfish communities. Along two different stretches of eroding shoreline, we created replicated pairs of subtidal breakwater reefs and established unaltered reference areas as controls. At both sites we measured shoreline and bathymetric change and quantified oyster recruitment, fish and mobile macro-invertebrate abundances. Breakwater reef treatments mitigated shoreline retreat by more than 40% at one site, but overall vegetation retreat and erosion rates were high across all treatments and at both sites. Oyster settlement and subsequent survival were observed at both sites, with mean adult densities reaching more than eighty oysters m(-2) at one site. We found the corridor between intertidal marsh and oyster reef breakwaters supported higher abundances and different communities of fishes than control plots without oyster reef habitat. Among the fishes and mobile invertebrates that appeared to be strongly enhanced were several economically-important species. Blue crabs (Callinectes sapidus) were the most clearly enhanced (+297%) by the presence of breakwater reefs, while red drum (Sciaenops ocellatus) (+108%), spotted seatrout (Cynoscion nebulosus) (+88%) and flounder (Paralichthys sp.) (+79%) also benefited. Although the vertical relief of the breakwater reefs was reduced over the course of our study and this compromised the shoreline protection capacity, the observed habitat value demonstrates ecological justification for future, more robust shoreline protection projects.
Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which ...is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body.
Potassium channels play an important role in electrical signaling of excitable cells such as neurons, cardiac myocytes, and vascular smooth muscle cells (VSMCs). In particular, the KCNQ (Kv7) family ...of voltage-activated K(+) channels functions to stabilize negative resting membrane potentials and thereby opposes electrical excitability. Of the five known members of the mammalian Kv7 family, Kv7.1 was originally recognized for its role in cardiac myocytes, where it contributes to repolarization of the cardiac action potential. Kv7.2 to Kv7.5 were first discovered in neurons, in which they play a well characterized role in neurotransmitter-stimulated action potential firing. Over the past 5 years, important new roles for Kv7 channels have been identified. Kv7 channels have been found to be expressed in VSMCs from several vascular beds where they contribute to the regulation of vascular tone. There is evidence that Kv7.5 channels in VSMCs are targeted by the hormone vasopressin to mediate its physiological vasoconstrictor actions and evidence that neuronal Kv7 channels in the baroreceptors of the aortic arch adjust the sensitivity of the mechanosensitive neurons to changes in arterial blood pressure. These newly identified physiological roles for Kv7 channels in the cardiovascular system warrant increased attention because pharmacological modulators of this family of channels are being used clinically to treat a variety of neurological disorders. This raises questions about the cardiovascular side effects associated with existing therapies, but there is also obvious potential to capitalize on the established and evolving pharmacology of these channels to develop new therapies for cardiovascular diseases.
Smooth muscle cells of the vasculature, viscera, and lungs generally express multiple α-subunits of the Kv7 voltage-gated potassium channel family, with increasing evidence that both Kv7.4 and Kv7.5 ...can conduct "M-currents" that are functionally important for the regulation of smooth muscle contractility. Although expression systems demonstrate that functional channels can form as homomeric tetramers of either Kv7.4 or Kv7.5 α-subunits, there is evidence that heteromeric channel complexes, containing some combination of Kv7.4 and Kv7.5 α-subunits, may represent the predominant configuration natively expressed in some arterial myocytes, such as rat mesenteric artery smooth muscle cells (MASMCs). Our previous work has suggested that Kv7.4/Kv7.5 heteromers can be distinguished from Kv7.4 or Kv7.5 homomers based on their biophysical, regulatory, and pharmacological characteristics, but it remains to be determined how Kv7.4 and Kv7.5 α-subunits combine to produce these distinct characteristics. In the present study, we constructed concatenated dimers or tetramers of Kv7.4 and Kv7.5 α-subunits and expressed them in a smooth muscle cell line to determine if a particular α-subunit configuration can exhibit the features previously reported for natively expressed Kv7 currents in MASMCs. Several unique characteristics of native smooth muscle M-currents were reproduced under conditions that constrain channel formation to a Kv7.4:Kv7.5 stoichiometry of 2:2, with alternating Kv7.4 and Kv7.5 α-subunits within a tetrameric structure. Although other subunit arrangements/combinations are not ruled out, the findings provide new insights into the oligomerization of α-subunits and the ways in which Kv7.4/Kv7.5 subunit assembly can affect smooth muscle signal transduction and pharmacological responses to Kv7 channel modulating drugs.
Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different ...sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5
-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP
) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to
voltage-sensing phosphatase (Ci-VSP)-induced PIP
depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP
, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP
affinity; these changes in PIP
affinity have corresponding effects on channel activity. Resting affinities for PIP
differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.
Halodule wrightii
(shoal grass) is a dioecious seagrass with a widespread tropical and subtropical distribution. Like all seagrass species,
H. wrightii
has the ability to expand asexually through ...rhizome elongation and to reproduce sexually through seed. To better understand
H. wrightii
sexual recruitment dynamics in the northern Gulf of Mexico, we investigated seed bank densities at 815 sites from south Texas to the Florida Panhandle.
H. wrightii
seed reserves were spatially variable across the region, with seed densities ranging from 0 to 5290 seeds m
−2
. Spatial analysis revealed clusters of high seed densities (“hot spots”) in Upper Laguna Madre, TX, and Santa Rosa Sound, FL, and clusters of low seed densities (“cold spots”) in Lower Laguna Madre and Aransas Bay, TX. Hot spots were dominated by
H. wrightii
, whereas cold spots were dominated by
Thalassia testudinum
(turtle grass). We frequently found intact seed coat halves, suggesting germination; however, we also encountered broken seed coat pieces, characteristic of seed predation. Genotypic surveys within and adjacent to seed hot spots revealed genetically diverse adult populations 6 years post seed bank sampling. Our data show that
H. wrightii
seed reserves are heterogeneous across the northern Gulf of Mexico and that the factors driving variation in seed bank density, viability, and germination remain poorly understood. Information on the spatial heterogeneity of
H. wrightii
seed densities has relevance for seagrass management, including targeting meadows with high levels of reproductive effort for protection or designation as marine reserves.
β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully ...elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.
Potassium channels are recognized as important regulators of cellular functions in most, if not all cell types. These cellular proteins assemble to form gated pores in the plasma membrane, which ...serve to regulate the flow of potassium ions (K+) from the cytosol to the extracellular space. In VSMCs, the open state of potassium channels enables the efflux of K+ and thereby establishes a negative resting voltage across the plasma membrane that inhibits the opening of VSCCs. Under these conditions, cytosolic Ca2+ concentrations are relatively low and Ca2+‐dependent contraction is inhibited. Recent research has identified Kv7 family potassium channels as important contributors to resting membrane voltage in VSMCs, with much of the research focusing on the effects of drugs that specifically activate or block these channels to produce corresponding effects on VSMC contraction and vascular tone. Increasingly, evidence is emerging that these channels are not just good drug targets—they are also essential intermediates in vascular signal transduction, mediating vasoconstrictor or vasodilator responses to a variety of physiological stimuli. This review will summarize recent research findings that support a crucial function of Kv7 channels in both positive (vasoconstrictive) and negative (vasorelaxant) regulation of microvascular tone.
Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that ...direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase cAMP (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.
Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, are important for the physiological maintenance of blood ...pressure, and they are also the basis for therapeutic use of vasopressin to restore blood pressure in hypotensive patients. However, the mechanisms by which circulating AVP induces arterial constriction are unclear. We examined the novel hypothesis that KCNQ potassium channels mediate the physiological vasoconstrictor actions of AVP. Reverse transcriptase polymerase chain reaction revealed expression of KCNQ1, KCNQ4, and KCNQ5 in rat mesenteric artery smooth muscle cells (MASMCs). Whole-cell perforated patch recordings of voltage-sensitive K+ (Kv) currents in freshly isolated MASMCs revealed 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one (linopirdine)- and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991)-sensitive KCNQ currents that were electrophysiologically and pharmacologically distinct from other Kv currents. Suppression of KCNQ currents by AVP (100 pM) was associated with significant membrane depolarization, and it was abolished by the protein kinase C (PKC) inhibitor calphostin C (250 nM). The KCNQ channel blocker linopirdine (10 microM) inhibited KCNQ currents in MASMCs, and it induced constriction of isolated rat mesenteric arteries. The vasoconstrictor responses were not additive when combined with 30 pM AVP, and they were prevented by the L-type Ca2+ channel blocker verapamil. Ethyl-N-2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl carbamic acid (flupirtine) significantly enhanced KCNQ currents, and it reversed constrictor responses to 30 pM AVP. In vivo, i.v. administration of linopirdine induced a dose-dependent increase in mesenteric artery resistance and blood pressure, whereas flupirtine had the opposite effects. We conclude that physiological concentrations of AVP induce mesenteric artery constriction via PKC-dependent suppression of KCNQ currents and L-type Ca2+ channel activation in MASMCs.
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