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First published online July 20, 2006
Journal of Experimental Biology 209, 2979-2989 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02314

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Cloning and functional expression of voltage-gated ion channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis

C. Bouchard¹, R. B. Price¹, C. G. Moneypenny¹, L. F. Thompson¹, M. Zillhardt¹, L. Stalheim¹ and P. A. V. Anderson^1,2,*

¹ Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd, St Augustine, FL 32080, USA
² Department of Physiology and Functional Genomics, University of Florida, 9505 Ocean Shore Blvd, St Augustine, FL 32080, USA

View larger version (51K): [in a new window]   Fig. 1. Micrographs of cnidocytes isolated from the tentacles of Physalia physalis. (A) A low power micrograph showing the absence of other cell types. (B) A combined Normarski/fluorescence micrograph showing representatives of the two size classes of cnidocytes present in these preparations. Blue fluorescence of DAPI marks the nucleus of the cells. (C,D) Higher power Normarski/fluorescence micrographs of a single large (C) and small (D) cnidocyte. The DAPI-stained nucleus is very evident at the base of the cyst in each cell. The cnidocil at the apical end of each cell is marked with an arrow. Note the cytoplasmic projections at the base of the cells. Scale bars: 200 µm (A), 50 µm (B), 20 µm (C,D).

View larger version (86K): [in a new window]   Fig. 2. An alignment of the amino acid sequence of the Physalia ß subunit (PpCaVß) (GenBank Accession number: ABD59026) with ß subunits from the scyphozoan jellyfish Cyanea capillata (CyCaVß [Accession Number: AAB87751]), one each from the platyhelminth Schistosoma mansoni (SmCaVß2 [AAK51118]), Drosophila (DmCaVß [AAF21096]), rabbit (CaVß1 [P19517]) and human (CaVß2 [AAL73495]). Conservation (100%) is indicated by white letters on a black background and greater than 80% conservation by white letters on a gray background. Residues that interact directly with the AID domain of the 1 subunit have a red background. This alignment was obtained using ClustalX (Version 1.83) and displayed using Genedoc (Vers. 2.6.002).

View larger version (17K): [in a new window]   Fig. 3. An unrooted phylogenetic tree showing the relationship of PpCaVß (shown in bold type) to other ß-subunits. Subunits included in this alignment are CyCaVß from the scyphozoan jellyfish Cyanea capillata [GenBank Accession number AAB87751], SmCaVß1 [AAK51117] and SmCaVß2 [AAK51118] from the platyhelminth Schistosoma mansoni, DmCaVß [AAF21096] from Drosophila, MdCaVß [A54844] from the fly Musca, and eight members of vertebrate ß subunits subfamilies found in rabbits (CaVß2 [P54288]); rat (CaVß1[P54283], CaVß2[Q8VGC3] and CaVß3[P54287]), and human (CaVß1 [Q02641], CaVß2 [AAL73495], CaVß3 [BAA07803], CaVß4 [O00305]). Only the bipartitions supported at greater than 95% probability are shown. Scale bar represents the average number of replacements per site.

View larger version (23K): [in a new window]   Fig. 4. Functional expression of PpCaVß. (A) Current/voltage relationships of currents gated by CyCaV in the presence (triangles) and absence (squares) of PpCaVß. Values are means ± s.d. of from 13 (triangles) to 16 (squares) trials. (B) Peak amplitude (± s.d.) of currents gated by CyCaV in the presence (black bar; N=13) and absence (white bar; N=16) of PpCaVß. In the presence of PpCaVß, currents are significantly larger (P<2x10-6). (C) Same, for currents gated by CaV2.3 in the presence (dark bar; N=14) and absence (white bar; N=14) of PpCaVß. In the presence of PpCaVß currents are significantly larger (P<0.012). (D) Current/voltage relationship of normalized currents (± s.d.) gated by CyCaV in the presence (triangles; N=13) and absence (squares; N=16) of PpCaVß. Co-expression with PpCaVß results in an obvious negative shift in the I/V relationship. (E) The same, for currents gated by CaV2.3 in the presence (triangles; N=16) and absence (squares; N=14) of PpCaVß. The I/V relationship of CaV2.3 is altered slightly by co-expression with PpCaVß. Data points in D and E are fitted by the Boltzman function Inorn=gnorm(V-Vrev)/(1+exp[-(V-V)]/k, where g=conductance, Vrev=53.44, 63.4, 66.25 and 68.94, V=9.68, -0.5652, -3.067 and -7.281 and k=9.443, 7.152, 7.357, and 6.163 for CyCaV, CyCaV/PpCaVß, CaV2.3 and CaV2.3/PpCaVß. (F) The relationship between time constant of inactivation of currents in the presence (triangles; N=14) and absence (squares; N=9) of PpCaVß. The time constant of inactivation was determined by fits of exponential curves to the decay phase of the current. At +10 mV, the time constant of inactivation of the current is significantly faster (P>0.0015) in the presence of PpCaVß. (G) Same, for currents gated by CaV2.3. There was no significant difference in the rate of inactivation under the two conditions.

View larger version (111K): [in a new window]   Fig. 5. Alignment of the Physalia K+ channel (GenBank Accession number: ABD59027), with other KV1 channels including two from jellyfish (jShak1 [Accession number AAB02603] and jShak2 [AAB02604]), the Drosophila Shaker channel (DmKV1 [P08510]), rabbit (KV1.4 [CAB94846]) and human (KV1.5 [NP 002225]). The degree of conservation is indicated as for Fig. 2. The transmembrane segments and pore regions are marked. This alignment was obtained using ClustalX and displayed using Genedoc (Ver. 2.6.002).

View larger version (14K): [in a new window]   Fig. 6. An unrooted phylogram showing the relationship between PpKV1 (shown in bold type) and other voltage-gated K+ channels. Channels used in this alignment include jShak1 [AAB02603] and jShak2 [AAB02604] from the hydromedusa Polyorchis pennicilatus, the Shaker (DmKV1 [P08510]), Shaw (DmKV3 [P17972]) and Shal (DmKV4 [P17971]) channels from Drosophila, one each from the squid, Loligo pealei (LpKV2 [CAA74748]) and tunicate Halocynthia roretzi (HrKV2 [BAA75810], and six mammalian channels, the human KV1.5 [AAH99666] and KV1.7 [NP 114092] channels, the mouse KV1.4 [NP 067250], KV3.1 [NP 032447] and KV3.2 [NP 001020752] channels, and rabbit KV2.2 [Q95L11] channel. Only the bipartitions supported at greater than 95% probability are shown. Scale bar represents the average number of replacements per site.

View larger version (16K): [in a new window]   Fig. 7. Functional properties of expressed PpKV1 channels. (A) Family of currents recorded from an oocyte under voltage clamp. (B) Current/voltage relationship of the currents shown in A. (C) An H-infinity plot showing the voltage-dependance of channel inactivation. 50% inactivation occurs at -19 mV.