Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca2+ channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca2+ channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca2+ channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca2+ channel α1H-subunit resulted in the generation of transient Ca2+ currents. Overnight treatment of α1H-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca2+ channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca2+ channels. Trafficking of α1H-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α1H-GFP subunits. Trafficking of T-type Ca2+ channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca2+ channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca2+ channels

Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties

Voltage-dependent calcium (Ca2+, CaV1.2) channels are the primary Ca2+ entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified CaV1.2 α1-subunit splice variation in myocytes of rat resistance-size (100–200 μm diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and ± 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major CaV1.2 variant contained exons 1c, 8, 21, and 32+33, with ∼57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ ± 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the CaV1.2 α1-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell CaV1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range