Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca 2+ -activated K + channels (BK Ca ) provides an opposing hyperpolarizing influence reducing Ca 2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K + currents were determined at pipette [Ca 2+ ] of 100 nm or 5 μm in the presence and absence of the BK Ca inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca 2+ levels. At 5 μm Ca 2+ , cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF −1 at +70 mV). Studies with IBTX suggested that the differences in K + conductance at 5 μm intracellular [Ca 2+ ] were largely due to activity of BK Ca . 17β-Oestradiol (1 μm), reported to potentiate BK Ca current via the channel's β-subunit, caused a greater effect on whole cell K + currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α-subunit-selective BK Ca opener, NS-1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca 2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BK Ca protein and the β 1: α-subunit ratio. The data support vascular heterogeneity with respect to the activity of BK Ca in terms of both β-subunit regulation and interaction with SR-mediated Ca 2+ signalling.

L-type, voltage-gated Ca2؉ channels (Ca L ) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle Ca L channel isoforms are acutely potentiated following ␣5␤1 integrin activation. Only the ␣ 1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by ␣5␤1 integrin requires phosphorylation of ␣ 1C C-terminal residues Ser 1901 and Tyr 2122 . These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following ␣5␤1 integrin activation, native Ca L channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of Ca L channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.Voltage-gated calcium channels play critical roles in the regulation of calcium entry across the plasma membranes of excitable cells. L-type calcium channels (Ca L ), 5 which are highly expressed in brain and muscle, are heteromeric transmembrane proteins composed of a poreforming ␣ 1C (Cav1.2) subunit along with accessory ␤, ␣ 2 , ␦, and sometimes ␥ subunits (1, 2). The ␣ 1C subunit contains four highly conserved repeat regions with 24 membrane-spanning domains, in addition to a variable length N terminus and relatively long, intracellular C terminus. The three ␣ 1C isoforms (neuronal, Cav1.2c; smooth muscle, Cav1.2b; cardiac, Cav1.2a) exhibit significant sequence differences in their N and C termini but all are regulated by intracellular kinases in ways that uniquely determine calcium entry and cell excitability.The regulation of Ca L channels by serine-threonine kinases has been extensively investigated. PKG phosphorylates a conserved serine reside in the cytoplasmic I-II linker (3) of all three ␣ 1C isoforms, leading to inhibition of current. PKC phosphorylates N-terminal threonine residues in cardiac and smooth muscle isoforms (4 -6) leading in most cases to potentiation of current. PKA phosphorylates all three ␣ 1C isoforms at a conserved C-terminal serine (Ser 1901 in Cav1.2c; Ser 1928 in Cav1.2a), thereby mediating ␤-adrenergic potentiation of the calcium current in cardiac myocytes and neurons (7-9). PKA also regulates ␣ 1C in smooth muscle, but the functional consequences on calcium current are complicated by crossover activation of PKG, which is expressed at high levels in that tissue (10).We recently demonstrated that Ca L currents in vascular smooth muscle (VSM) are acutely regulated by the integrin class of cel...

Ion channels are regulated by protein phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues. Evidence for the latter process, tyrosine phosphorylation, has increased substantially since this topic was last reviewed. In this review, we present a comprehensive summary and synthesis of the literature regarding the mechanism and function of ion channel regulation by protein tyrosine kinases and phosphatases. Coverage includes the majority of voltage-gated, ligand-gated, and second messenger-gated channels as well as several types of channels that have not yet been cloned, including store-operated Ca2+ channels, nonselective cation channels, and epithelial Na+ and Cl- channels. Additionally, we discuss the critical roles that channel-associated scaffolding proteins may play in localizing protein tyrosine kinases and phosphatases to the vicinity of ion channels.

Lymphatic collecting vessels exhibit spontaneous contractions with a pressure-dependent contraction frequency. The initiation of contraction has been proposed to be mediated by the activity of a Ca2+-activated Cl− channel (CaCC). Here, we show that the canonical CaCC Anoctamin 1 (Ano1, TMEM16a) plays an important role in lymphatic smooth muscle pacemaking. We find that isolated murine lymphatic muscle cells express Ano1, and demonstrate functional CaCC currents that can be inhibited by the Ano1 inhibitor benzbromarone. These currents are absent in lymphatic muscle cells from Cre transgenic mouse lines targeted for Ano1 genetic deletion in smooth muscle. We additionally show that loss of functional Ano1 in murine inguinal-axillary lymphatic vessels, whether through genetic manipulation or pharmacological inhibition, results in an impairment of the pressure–frequency relationship that is attributable to a hyperpolarized resting membrane potential and a significantly depressed diastolic depolarization rate preceding each action potential. These changes are accompanied by alterations in action potential shape and duration, and a reduced duration but increased amplitude of the action potential–induced global “Ca2+ flashes” that precede lymphatic contractions. These findings suggest that an excitatory Cl− current provided by Ano1 is critical for mediating the pressure-sensitive contractile response and is a major component of the murine lymphatic action potential.

the spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. these contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. in previous studies, t-type voltage-gated ca 2+ channels (VGccs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and ni 2+. Our goal was to test this idea in a more definitive way using genetic knock out mice. first, we demonstrated through both pcR and immunostaining that mouse lymphatic muscle cells expressed ca v 3.1 and Ca v 3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each t-type VGcc isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. further, both Wt and ca v 3.1 −/− ; 3.2 −/− double knockout lymphatic vessels responded similarly to mibefradil and ni 2+ , which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than t-type VGccs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and ca v 3.1 −/− ; 3.2 −/− double knockout mice. in contrast, smoothmuscle specific deletion of the L-type VGCC, Ca v 1.2, completely abolished all lymphatic spontaneous contractions. collectively our results suggest that, although t-type VGccs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and ni 2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions. The spontaneous contractions of collecting lymphatic vessels propel lymph centrally to account for 2/3 of peripheral lymph flow 1,2. These rapid, large-amplitude contractions are analogous to twitch contractions of cardiac and skeletal muscle and are particularly important for moving lymph uphill against the adverse hydrostatic gradients that exist in dependent extremities. Lymphatic contractions are triggered by action potentials (APs) in lymphatic smooth muscle cell...