Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Abstract

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.

Fig. 1.

Expression of CaM-sensor MLCK in A7r5 smooth muscle cells. (A) Schematic representation illustrating change in structure of CaM-sensor MLCK upon Ca²⁺/CaM binding. Ellipsoids, with solved (CaM, light blue) and modeled (MLCK, magenta) ribbon structures inserted, represent structures determined previously by neutron scattering (27). EYFP and ECFP are shown as yellow and cyan cylinders linked by the CaM-binding sequence (red) and located C-terminal to the native CaM-binding sequence (red). When Ca²⁺/CaM binds to the two CaM-binding sequences, it displaces the autoinhibitory sequence (dark blue line) to expose the catalytic cleft (dark blue circle) and separates the EYFP/ECFP dimer, resulting in diminished FRET. (B) Rat thoracic aortic A7r5 cells transfected with pSM8 35 KCS plasmid. CaM-sensor MLCK bound to stress fibers was visualized 72 h after transfection by fluorescence microscopy. (C) Western blots of lysates and immunoprecipitates from A7r5 cells. Cells were transfected with pSM8 35 KCS plasmid and lysates collected after 72 h. Proteins were immunoprecipitated with either K36 or anti-GFP Abs and analyzed by Western blotting using K36 mAb to detect MLCK (short and long isoforms) endogenous to A7r5 cells and CaM-sensor MLCK. (D) MLCKs immunoprecipitated with anti-GFP Abs from lysates of A7r5 cells expressing CaM-sensor MLCK or MLCK fused to GFP show Ca²⁺-dependent activity in the presence of CaM.

Fig. 2.

Expression of CaM-sensor MLCK in smooth muscle tissues of pSM8 35 KCS transgenic mice. (A) Western blot analysis of tissue extracts from transgenic and control mice with anti-MLCK Ab K36. Upper arrow, CaM-sensor MLCK; lower arrow, short MLCK. (B) Confocal images of a region of myocardium from transgenic mice containing a blood vessel. GFP was detected with Rho-labeled secondary Ab, whereas GFP fluorescence was detected in the fluorescein channel. (C) Images of control (Left) and CaM-sensor MLCK transgenic (Right) urinary bladder in situ. Photographs were taken with a stereo microscope equipped with MAA-03 Universal Light Source (BLS, Budapest) for GFP visualization (Upper) and standard illumination (Lower). (D) Confocal images of bladder cross-section from CaM-sensor transgenic mice. Tissue sections were treated as in B.

Fig. 3.

Fluorescent properties of permeabilized bladder strips. (A) Emission spectrum of Triton X-100-permeabilized bladder smooth muscle from CaM-sensor MLCK transgenic mice at pCa 8 to 3.7. Measurement of CaM-sensor MLCK FRET ratio (B) and force (C) in Triton X-100-permeabilized strip.

Fig. 4.

Simultaneous measurements of force and intracellular calcium (Upper) or force and FRET (Lower) in intact bladder strips stimulated by depolarization (KCl) or carbachol (CCh).

Fig. 5.

Measurements of [Ca²⁺]_i, MLCK activation, RLC phosphorylation and force in intact bladder strips. Average values are shown for control (PSS), or contracted strips treated for 1 min with 65 mM KCl (KCl), 10 μM carbachol (CCh), or a mixture of KCl and CCh in PSS containing 10 mM calcium (KCl/Ca/CCh). Values represent mean ± SEM (n ≥ 5). ^*, P < 0.05, compared with PSS group; #, P < 0.5, compared with KCl group.