Photochemical gating of intracellular Ca2+ release channels

Article abstract of DOI:10.1021/ja069361q

We have synthesized BiNiX, a caged methylxanthine agonist for the ryanodine receptor (RyR) - the major calcium channel that mediates Ca²⁺ release from intracellular Ca²⁺ stores in electrically excitable cells. BiNiX is easily loaded

Full text of DOI:10.1021/ja069361q

Published on Web 04/11/2007
Photochemical Gating of Intracellular Ca2+ Release Channels
Jiahong Ni,† Darryl A. Auston,†,‡ David A. Freilich,†,§ Sukumaran Muralidharan,† Eric A. Sobie,| and
Joseph P. Y. Kao*,†,‡,§
Medical Biotechnology Center, UniVersity of Maryland Biotechnology Institute, Baltimore, Maryland 21201,
Department of Physiology and Program in Neuroscience, UniVersity of Maryland School of Medicine,
Baltimore, Maryland 21201, and Department of Pharmacology and Biological Chemistry,
Mount Sinai School of Medicine, New York, New York 10029
Received December 29, 2006; E-mail: jkao@umaryland.edu
Chart 1. Methyxanthines That Activate Ryanodine Receptors
Electrically excitable cells abundantly express a channel that
mediates release of Ca2+ from intracellular calcium stores. These
release channels are blocked by the alkaloid, ryanodine, and are
therefore called ryanodine receptors (RyRs).1 In nerve and muscle
cells, electrical excitation of the cell membrane opens voltage-
dependent Ca2+ channels, which conduct Ca2+ ions from the
extracellular solution into the cytosol. In turn, these Ca2+ ions can
bind to, and thus trigger the opening of, RyRs to permit massive
Ca2+ flux from intracellular stores into the cytosol to raise the
cytosolic free Ca2+ concentration ([Ca2+]i). This process is known
Scheme 1. Synthesis of BiNiX and Its Acetoxymethyl Estera
as “Ca2+-induced Ca2+ release” (CICR). The increased [Ca2+
]
i
consequent to CICR is crucial to cardiac muscle contraction.2 CICR
can also regulate electrical excitability in neurons by controlling
the opening of Ca2+-activated K+ channels.3 In the course of our
research on neuronal excitability, we wished to open RyRs rapidly
without activating the voltage-dependent Ca2+ influx that normally
serves as a trigger. To this end, we designed and synthesized a
“caged” agonist that, when photolyzed intracellularly, activates
RyRs rapidly and robustly.
A “caged” molecule is an inert but photolabile molecule that is
transformed by photolysis into a biologically active molecule; this
process is often called “uncaging” or “photorelease”.4 Because the
spatial resolution of photorelease is limited only by the ability to
focus light, diffraction-limited (sub-micrometer) resolution is
potentially achievable. Moreover, fast photochemical kinetics enable
fast photorelease. Therefore, focal photolysis of caged molecules
is an excellent tool for controlling biological processes with high
spatiotemporal precision. The most common approach to caged
molecules is to attach a photolabile protective group to an essential
functional group in a biomolecule and thus abolish its biological
activity. Photolytic cleavage of the protective group unmasks the
functional group to restore bioactivity.
Methylxanthines, such as theophylline and caffeine, are RyR
agonists (Chart 1).5 They bind to RyRs and induce channel opening
even at resting levels of [Ca2+]i (e0.1 µM). As a starting point,
we sought a methylxanthine that has (1) good potency and efficacy
for activating RyRs, (2) high water solubility, (3) high membrane
permeancy to permit easy passage through cell membranes, and
(4) accessibility to chemical modification. Paraxanthine (PX; 1,7-
dimethylxanthine) uniquely satisfied these criteria.5 As a photolabile
protective group, we used 4,5-bis(carboxymethoxy)-2-nitrobenzyl
(BcmNb), which, when attached to the 3 position of PX, would
yield a highly water-soluble caged molecule bearing two carboxylate
groups (3-BcmNb-PX, or BiNiX for brevity). Finally, we wished
to mask the carboxylates on the caged molecule as acetoxymethyl
a Reagents and conditions: (a) BrCH2CO2Et, K2CO3, DMF, 0 °C to room
temp; (b) KNO3, TFA, 0 °C to room temp; (c) NaBH4, CH2Cl2/EtOH/HOAc
(35:5:1), 0-5 °C; (d) MsCl, Et3N, CH2Cl2, 0 °C to room temp; (e) 1,7-
dimethylxanthine, K2CO3, n-Bu4NHSO4, DMF, room temp; (f) (i) 1 N
NaOH, CH2Cl2/MeOH (1:1), room temp; (ii) 1 N HCl; (g) BrCH2OAc,
Et3N, DMF, room temp.
(AM) esters, which are labile to cleavage by intracellular esterases.6
The AM ester, being lipophilic and thus membrane-permeant, can
enter a cell by diffusion across the cell membrane. Thereafter,
intracellular esterases rapidly cleave the AM esters to expose the
carboxylates on BiNiX, which, being a dianion at physiologic pH,
cannot readily cross the cell membrane, and is therefore trapped
inside the cell. Thus, BiNiX can be accumulated intracellularly.
The synthesis of BiNiX and the corresponding AM ester is
outlined in Scheme 1. The reagent for introducing the BcmNb
protective group was prepared from 3,4-dihydroxybenzaldehyde in
four steps: O-alkylation with ethyl bromoacetate, nitration, reduc-
tion of the aldehyde to the benzylic alcohol, which was then
converted to the mesylate. Alkylation of paraxanthine with the
mesylate, followed by hydrolysis, yielded water-soluble BiNiX.
Reaction of BiNiX with bromomethyl acetate afforded the lipophilic
AM ester.
In aqueous solution, BiNiX has a near-UV absorption peak at
λmax ) 346 nm (ꢀ ) 5.72 × 103 M-1 cm-1; spectrum in Supporting
Information). Photolysis of BiNiX in sodium phosphate buffer (0.1
M, pH 7.4) at 355 nm proceeded with a quantum efficiency of φ
) 0.0058 ( 0.0002. To study the kinetics of photolysis, we used
transient absorption spectroscopy to monitor the photolytically
generated, short-lived aci-nitro intermediate,7 whose decay is
concomitant with cleavage of the protective group and release of
product.8 The transient absorption decay followed a single-
exponential time course, with a time constant of τ ) 0.26 µs (or
t1/2 ) 0.18 µs; see Supporting Information). Thus, flash photolysis
† University of Maryland Biotechnology Institute.
‡ Department of Physiology, University of Maryland School of Medicine.
§ Program in Neuroscience, University of Maryland School of Medicine.
| Mount Sinai School of Medicine.
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10.1021/ja069361q CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
was amplified regeneratively by CICR into a wave of elevated
[Ca2+]i that swept through the cell. The Ca2+ wave triggered
contraction of the myocyte, as illustrated by the length-versus-time
trace in Figure 1 (a time-lapse video of the experiment is available
as Supporting Information). The light-evoked Ca2+ release was
completely abolished in the presence of 10 µM ryanodine, a specific
inhibitor of RyRs. In cells loaded with Fluo-3 but not BiNiX,
identical light flashes evoked no response (figure in Supporting
Information). These findings indicate that the observed response
was not artifactual, but rather resulted from specific RyR activation.
Two examples illustrate the potential utility of BiNiX in cellular
physiology. First, gating of RyRs depends on both cytosolic and
SR luminal [Ca2+]. Activating RyRs with photoreleased PX, instead
of a cytosolic [Ca2+] rise, allows the regulation by SR luminal
[Ca2+] to be probed independently. This is important for studying
spontaneous Ca2+ waves that can trigger cardiac arrhythmias.
Second, Ca2+ released through RyRs can profoundly affect ion
channels and electrical excitability in neurons; however, little is
known about the spatial distribution of RyRs in these anatomically
complex cells. In conjunction with fluorescent Ca2+ indicators such
as Fluo-3, focal photorelease of PX affords a means for mapping
the distribution of RyRs in neurons.
In summary, we have designed, synthesized, and demonstrated
the biological utility of BiNiX, a photoactivatable RyR agonist.
Through incubation with the AM ester form, BiNiX can be easily
loaded into cells. Thereafter, focal photolysis permits activation of
RyRs with high spatiotemporal precision. Therefore, BiNiX is a
unique tool for selectively probing RyR function in living cells.
Acknowledgment. We thank Mr. Marcus Palmer for preparing
the cardiac myocytes. This research was supported by the NIH
(Grants GM056481 and GM64706 to J.P.Y.K.).
Supporting Information Available: Details of chemical syntheses,
physical chemical measurements, biological experiments, and charac-
terization data for all intermediates; video (avi). This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 1. In a cardiac myocyte, subcellular photorelease of paraxanthine
in a 5-µm spot (white circle) triggers a Ca2+ wave and consequent
contraction of the myocyte (yellow scale bar ) 10 µm). An 8-ms flash of
355-nm light from a quasi-continuous Nd:YAG laser was delivered
immediately before acquiring the image at 0 ms. Intracellular [Ca2+] was
monitored by imaging Fluo-3 fluorescence. Fluorescence intensity is encoded
in pseudocolor according to the color-scale bar. Changes of fluorescence
and cell length relative to baseline are shown as red and blue line traces,
respectively.
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