A junctophilin-caveolin interaction enables efficient coupling between ryanodine receptors and BKCa channels in the Ca2+ microdomain of vascular smooth muscle.

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-ß-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.

Conversion of Ca2+ oscillation into propagative electrical signals by Ca2+-activated ion channels and connexin as a reconstituted Ca2+ clock model for the pacemaker activity.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl- channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18ß-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

The multiple expression of Ca²âº-activated Cl⁻ channels via homo- and hetero-dimer formation of TMEM16A splicing variants in murine portal vein.

Ca(2+)-activated Cl(-) channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments ("a" and "b" in N-terminus and "c" and "d" in the first intracellular loop), were identified; the expression ratio of four transcripts of "abc", "acd", "abcd" and "ac" was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A(inh)-A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.