MODULATION OF NEURONAL ACTIVITY BY INTRACELLULAR MESSENGERS 
Steven A. Siegelbaum, Ph.D., Associate Investigator 
Dr. Siegelbaum has continued to focus on the 
molecular mechanism of ion channel modulation 
by second messengers in moUuscan and vertebrate 
neurons. In addition, the role that intracellular cal- 
cium plays in both the induction and expression of 
neuromodulation has been studied. 
Arachidonic acid and its lipoxygenase metabolites 
have recently been shown to serve as second mes- 
sengers in neurons. Previously this laboratory, in 
collaboration with Drs. Eric R. Kandel and James H. 
Schwartz (HHMI, Columbia University), showed 
that the 12-lipoxygenase metabolite of arachidonic 
acid, 12-HPETE (12-hydroperoxyeicosatetraenoic 
acid), mediated an increase in opening of the S-type 
channel of Aplysia sensory neurons in response 
to the neuropeptide FMRFamide (Phe-Met-Arg-Phe- 
amide). The increase in K""" current hyperpolarizes 
the neuron and contributes to presynaptic inhibi- 
tion. During the past year this work has been ex- 
tended by 1) investigating the mechanism whereby 
12-HPETE causes S channel opening and 2) explor- 
ing the potential role of arachidonic acid metabo- 
lites as modulators of neuronal function in chick 
sympathetic neurons. 
L Modulation of S K"*" Channel Activity 
by 12-HPETE. 
Four possible mechanisms by which 12-HPETE 
could modulate S channel activity include 1) pro- 
tein kinase activation, leading to S channel phos- 
phorylation; 2) phosphatase activation, leading to 
S channel dephosphorylation; 3) G protein activa- 
tion, leading to direct G protein gating of the S 
channel; and 4) a direct action of 12-HPETE on 
S channel activation. To investigate these possibili- 
ties, Ned Buttner, Dr. Andrea Volterra, and Dr. 
Siegelbaum applied various arachidonic acid metab- 
olites directly to cell-free patches of membrane that 
lack ATP or GTP The results of these experiments 
show that 12-HPETE causes a dramatic and revers- 
ible increase in S channel opening in both cell-free 
inside-out and outside-out patches, where the in- 
side or outside of the membrane faces the bathing 
solution. Moreover, this effect is specific for this 
particular metabolite, since 12-HETE (a stable 
breakdown product of 12-HPETE), arachidonic 
acid, or 15-HPETE have no effect on S channel ac- 
tivity. Since the bath contained neither ATP nor GTP, 
the effect of 12-HPETE cannot involve phosphoryla- 
tion or G protein activation, and since the effect is 
readily reversible, phosphatase activation is also 
ruled out. Therefore it was concluded that 12- 
HPETE (or possibly some downstream metabolite) 
modulates S channel activity by acting directly on 
the S channel or a protein closely linked to the 
S channel in the cell-free patches. Surprisingly, 12- 
HPETE was effective at 100-fold lower concentra- 
tions when applied to outside-out patches com- 
pared with its action on inside-out patches. This 
suggests that the 12-HPETE receptor may be local- 
ized to the outer surface of the S channel. 
IL Modulation of Calcium Channel Activity in Chick 
Sympathetic Neurons by Arachidonic Acid. 
Do the arachidonic acid metabolites also modu- 
late channel activity in vertebrate neurons? To ad- 
dress this question. Dr. Siegelbaum is studying the 
effects of arachidonic acid on calcium channel func- 
tion in chick sympathetic neurons, in collaboration 
with Dr. Lorna Role (Columbia University), Dr. 
Linda Simmons, and Bill Bug. Many transmitters 
and peptides have been shown to produce presyn- 
aptic inhibition in sensory neurons through cal- 
cium current inhibition; however, the mechanism 
of this inhibitory action is unknown. The experi- 
ments of these investigators s how that arachidonic 
acid (0.3-10 |jlM) exerts a powerful inhibitory effect 
on the calcium current. A related fatty acid with but 
a single double bond (eicosamonoenoic acid) has 
no effect on the calcium current. Preliminary char- 
acterization of the calcium current suggests that it 
corresponds to a high-voltage-activated, dihydro- 
pyridine-insensitive type of calcium current. 
III. Intracellular Calcium Modulation by Serotonin 
and FMRFamide. 
The third project concerns the control and mod- 
ulation of intracellular calcium concentration by 
neurotransmitters in Aplysia sensory neurons. Pre- 
vious studies have shown that serotonin (5-HT) 
produces presynaptic facilitation, and FMRFamide 
produces presynaptic inhibition. It has been pro- 
posed that these actions are due to a modulation in 
the size of the internal calcium transient in re- 
sponse to action potentials. Using the fluorescent 
Ca^^ indicator Fura-2 and digital video imaging, 
Hal Blumenfeld and Dr. Siegelbaum have investi- 
Continued 
537 
