(A) Sequence of squid CE cDNA (GenBank accession no. U49390U49390). The coding region is in boldface type. (B) Amino acid sequence of CE compared with Amphioxus SCP I (29), yeast sar1p (30), and arf1 (31). Amino acids found by peptide sequencing of the tryptic digest are underlined. (C) Possible GTP-binding sites in CE, compared with homologous regions in sar1p.

(A) Single-channel current traces of K⁺ channel activity in an excised inside-out membrane patch of human skin cultured fibroblast recorded in the absence of CE 7 min after excision of the membrane patch (middle trace), at 10 min after addition of heat-inactivated CE (bottom trace), and at 3 min after addition of intact CE (top trace). The channel shown was typical of those recorded at −40 mV. Pipette solution was 140 mM KCl/2 mM CaCl₂/1 mM MgCl₂/10 mM Hepes (NaOH)/0.001 mM tetrodotoxin, pH 7.4; bathing solution was 150 mM NaCl/5 mM KCl/2 mM CaCl₂/1 mM MgCl₂/10 mM Hepes (NaOH), pH 7.4. Addition of protein was made by a 20-μl plastic pipette near the tip of the patch micropipette as 10-μl aliquots of CE in 10 mM imidazole·HCl. Petri dish contained 2 ml of bath solution. CE markedly reduced the mean open time (τ_open) and probability of openings. (B) Amplitude distributions of unitary K⁺ current records were fitted with a Gaussian function. Unitary current peak amplitudes were −10.6, −11.0, and −9.9 pA after addition of CE and heat-inactivated CE, and no addition, respectively. There were no significant differences in distribution among the three groups. (C) Open time distributions for the data collected during 30-s periods after excision of the membrane (as mentioned in A) were fitted to a single exponential. Exponential decay constants for open time distribution were 1.4, 12.0, and 12.5 ms after addition of CE and heat-inactivated CE, and no addition, respectively. CE application markedly reduced the open times of the K⁺ channel. (D) Membrane excitability increases due to CE. (Left) Passively propagated somatic spikes (small amplitude) occurred spontaneously in most cells, including the one illustrated here, before CE injection (pre). Spontaneous local dendritic calcium spikes (large amplitude) did not occur in some cells (upper trace, pre-calexcitin) but then did occur after injection of CE (post) through the recording electrode. In Purkinje cells hyperpolarized 20 mV below the membrane potential for somatic spikes, calcium spikes could be elicited by lower levels of injected positive current pulses after injection of CE. (Right) Mean percent change in the threshold current required to elicit local calcium spikes was lower for CE-injected cells vs. controls injected with heat-inactivated CE or 3 M potassium acetate (P < 0.001, Student’s t test). (E) Intracellular recordings of the Hermissenda type B photoreceptor response to a 1-s flash of light (10³ erg/cm²·sec) before (Upper) and after (Lower) injection of purified cloned CE. The Hermissenda photoreceptors were isolated and submerged in artificial sea water (430 mM NaCl/10 mM KCl/10 mM CaCl₂/50 mM MgCl₂/10 mM Hepes Na, pH 7.4). The CE (intraelectrode concentration, 364 nM) was brought to 1 M in KAc (pH 7.4) and injected (3 min, 2 nA) into the photoreceptor with the recording electrode. Recordings were obtained using intracellular amplifier (Axopatch 2A), digitized at 50 Hz (Digidata 1200), and analyzed by computer. The normal light response returned to the original resting potential within a minute (Upper). Injection of CE (n = 5 cells) enhanced the light response and the cell remained depolarized for more than 5 min (Lower, only 1 min is represented). Injection of heat-inactivated CE (n = 6 cells) using the above protocol did not alter the light response. Time scale at right is compressed by 3×.

(A) Northern blot of squid CE mRNA. The positions of 18S and 28S rat rRNA are shown. CE mRNAs were approx. 9.99 and 8.17 kb long. (B Left) ⁴⁵Ca overlay blot of oligohistidine–CE fusion protein (M_r = 24,000). Binding was performed in the presence of Mg²⁺ and the absence of GTP. Numerous other control proteins did not bind Ca²⁺ under these conditions. The lower band is a proteolytic degradation product. (B Right) Polyclonal antibody stained Western blot of oligohistidine–CE fusion protein. (C) Polyclonal antibody stained Western blot of squid optic lobe homogenate. (D Left) [³²P]GTP overlay blot of GST–CE fusion protein (M_r ≈ 50,000). Binding was performed in the absence of Ca²⁺. In a separate experiment, no binding to [³²P]ATP could be detected. The lower band is a proteolytic degradation product. (D Right) Polyclonal antibody stained Western blot of GST–CE fusion protein. After thrombin treatment, the band was reduced to 22 kDa, comigrating with native CE (C). (E) Ca²⁺ binding as a function of CE concentration. CE (0.8 μM) was incubated with various concentrations of ⁴⁵Ca and filtered through a nitrocellulose membrane. ⁴⁵Ca bound to the membrane was measured by scintillation counting. (F) Scatchard Ca²⁺-binding plot. Ca²⁺ binding was measured in the presence of 50 mM Tris·HCl, pH 7.4/50 mM KCl/5 mM MgCl₂/0.01 mM EGTA. The dissociation constant was unchanged when measured in the absence of Mg²⁺. (G) Phosphorylation of cloned squid CE by α-PKC in the absence (upper curve) and presence (lower curve) of GTP. The K_m of PKC for CE was estimated at 5.0 nM. Under these conditions, GTP inhibited PKC phosphorylation of histones by less than 30%.

Immunohistochemical localization of CE in the squid optic lobe. Sections were incubated with CE antibody and visualized by immunoperoxidase staining. Nuclei were counterstained blue with hematoxylin (g.o., outer granule cell layer; g.i., inner granule cell layer; p, plexiform layer). Arrow points to an amacrine cell. The plexiform layer containing neuronal axons and branches is darkly stained with the immunolabel.