Effects of photolysis of 5cgY-RS-20 on Ca²⁺ current enhancement and shortening in Bufo marinus smooth muscle cells. (A) Voltage clamp protocol used to demonstrate enhancement of inward Ca²⁺ currents (I_Ca) by repetitive depolarization of single smooth muscle cells. (B and C) Currents during initial 3-sec depolarizations before (T₁) and 12 sec after the train (T₂) are shown for an experiment in which 200 μM 5cgY-RS-20 or 200 μM caged tyrosine (cgY) were included in the pipette. The protocol was carried out on each cell before (Left) and just after (Right) 15-sec exposure to a xenon source UG11 filtered to pass near-UV light. (D) Summary of the effect of 5cgY-RS-20 and cgY on enhancement of inward Ca²⁺ current. The mean (± SEM) peak inward current after versus before the conditioning train (T₂/T₁) is shown for seven and five cells with 5cgY-RS-20 and cgY, respectively. (E) Images of a smooth muscle cell attached to a pipette containing 200 μM 5cgY-RS-20 in the whole-cell configuration. Images were observed just before and 1 and 3 sec after depolarization with the train of pulses shown in A. (F) Same cell as in E but after illumination to release 5Y-RS-20. (G) Mean (± SEM) rates of shortening of four cells before and after photorelease of 5Y-RS-20.

Effects on MLCK activity and eosinophil locomotion of variants of MLCK inhibitory peptide LSM1. (A) Amino acid sequences of LSM1 and variants containing glutamate (E) and caged tyrosine (cgY). (B) Effects of LSM1 peptides on constitutively active catalytic domain of MLCK before (•) and after (○) photoconversion; the photoreleased peptide was indistinguishable from authentic LSM1 in inhibitory potency, chromatographic behavior and mass spectra. Loss of inhibitory potency for 9E-LSM1 (▵) illustrates the requirement for Y in position 9 for inhibitory activity. Data represent means of triplicate measurements with SEM values less than 10% of the mean. (C–J) Effects of photorelease of LSM1 on eosinophil locomotion. Phase contrast images of an eosinophil injected with 9cgY-LSM1, before (C and D), during (E), and after (F–H) near-UV exposure with times in sec. Color coded outlines of the same cell at the designated intervals before (I) and after (J) photorelease of LSM1.

(A) Amino acid sequences of calmodulin inhibitory peptide RS-20 and variants containing glutamate (E), tyrosine (Y), and caged tyrosine (cgY). Peptide and caged peptide structures were confirmed by amino acid composition and mass spectrometry (see Materials and Methods). (B) Effects of RS-20 peptides on Ca²⁺/calmodulin-dependent MLCK activity in vitro. 5cgY-RS-20 before (•) and after (○) photoconversion; the photoreleased peptide was indistinguishable from authentic 5Y-RS-20 in inhibitory potency, chromatographic behavior, and mass spectra. There was no detectable inhibition by 5E-RS-20 (▵), whereas 5,18cgY-RS-20 inhibited only weakly both before (▴) and after photolysis (▪). Data points represent means of triplicate measurements with SEM values less than 10% of the mean. (C) Structure and photolysis reaction of cgY.

Acute paralysis of locomotion after photorelease of the calmodulin inhibitory peptide from 5cgY-RS-20. (A–D) Phase contrast images before (−40 and −20) and after (+40, +80, and +120) near-UV exposure (0) with times in sec. (E and F) Color-coded outlines showing cell movements at 20-sec intervals during the 5-min control period (“con”) and immediately after release of the peptide (“pht”). The cell outline 5 min after photolysis was virtually identical to that 40–60 sec after photolysis. (G and H) Enlargements of boxed areas in D showing continued edge ruffling 70–93 sec after peptide release. (I-K) Phase contrast images and outlines of a control cell and its lamellipods at 20-sec intervals before (“con”) and after (“pht”) the same near-UV light exposure used in A-D.