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First published online August 31, 2004
Journal of Experimental Biology 207, 3581-3590 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01204

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Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays

M. L. Tierney^1,*, K. E. Osborn¹, P. J. Milburn², M. H. B. Stowell³ and S. M. Howitt¹

¹ School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia
² John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia
³ MCD Biology, University of Colorado, Boulder, CO 80309, USA

View larger version (18K): [in a new window]   Fig. 1. Synthesis of 4,7,10-trioxa-1,13-carboxyethyl-2-trimethylamine-tridecanediaamine (TDAC).

View larger version (71K): [in a new window]   Fig. 2. RT-PCR products generated using specific primers for acetylcholine receptor genes amplified from H. monopterigium. RNA isolated from the electric organ of the coffin ray was reverse transcribed and amplified using primers specific for either the , ß, or genes of the acetylcholine receptor. Two RNA concentrations were tested with each primer pair, and RT-PCR products were run on a 0.8% agarose gel and stained with ethidium bromide. Lambda DNA cut with EcoR1 and HindIII was used to estimate the size of the DNA fragments (left-hand lane).

View larger version (17K): [in a new window]   Fig. 3. Purification of the acetylcholine receptor from the numb fish N. tasmaniensis. (A) The membrane fraction from the electric organ was treated with 1.5% Triton X-100, and solubilized material applied to the TDAC affinity column. Bound protein was eluted from the column using an increasing salt gradient. (B) The major contaminant (98 kDa) could be purified away from the acetylcholine receptor on an anion exchange column (ResourceQ), where it eluted later in the NaCl gradient. (C) The purified receptor ran close to the void volume on a Superdex200 gel permeation column (670 kDa), consistent with the receptor maintaining its dimeric form under the purification conditions used. Arrows indicate the elution positions of molecular mass markers used to calibrate the column: 670, 158, 44, 17 and 1.35 kDa. Absorbance was measured at 215 nm in A and C and at 280 nm in B.

View larger version (57K): [in a new window]   Fig. 4. SDS–PAGE of the acetylcholine receptor purified from the numb fish N. tasmaniensis on the TDAC affinity column. Fractions 3, 5 and 7, correlating to the peak absorbance in Fig. 3A, were run on a 4–12% Bis-Tris NuPAGE gel and stained with Coomassie. The protein bands corresponding to the four subunits comprising the acetylcholine receptor are indicated on the right. The protein running between the and ß subunits is mostly likely the subunit, which is known to run as a doublet.

View larger version (22K): [in a new window]   Fig. 5. A rapid filtration method through DE-81 filters was used to measure the binding of 125I--bungarotoxin (-Bgt) to affinity-purified acetylcholine receptors from electric rays at equilibrium. The data presented are from a typical experiment where the specific binding is plotted against increasing -Bgt concentrations. The dissociation constants calculated from the equation for a single binding site were not significantly different in the four electric rays tested, ranging between 21 and 53 nmol l–1. Error bars represent S.E.M.

View larger version (119K): [in a new window]   Fig. 6. Electron microscopic images of purified acetylcholine receptors from electric rays show them to be dimers. The affinity-purified acetylcholine receptor from H. monopterigium was viewed under the electron microscope in negative stain and is shown in A. Receptor dimers are evident in this (boxes in A) and all other acetylcholine receptor preparations isolated on the TDAC affinity column: (B) T. marmorata; (C) N. tasmaniensis.