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First published online February 27, 2009
Journal of Experimental Biology 212, 761-767 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026971
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Expression of a poriferan potassium channel: insights into the evolution of ion channels in metazoans

Gabrielle J. Tompkins-MacDonald1, Warren J. Gallin1, Onur Sakarya2,*, Bernard Degnan3, Sally P. Leys1 and Linda M. Boland4,{dagger}

1 University of Alberta, Department of Biological Sciences, Edmonton, AB, Canada T6G 2E9
2 University of California, Neuroscience Research Institute, Santa Barbara, CA 93117, USA
3 School of Integrative Biology, University of Queensland, Brisbane 4072, Australia
4 University of Richmond, Department of Biology, Richmond, VA 23173, USA


Figure 1
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  		Fig. 1. LALIGN global alignment of predicted amino acid sequences of two sponge inward-rectifier potassium (Kir) channels, AmqKirA and AmqKirB, cloned from Amphimedon queenslandica. Membrane-spanning regions deduced from hydropathy analysis and comparison with vertebrate Kir channels are noted by bars (M1, M2). Putative protein kinase A (PKA) (•) and protein kinase C (PKC) ({blacktriangleup}) phosphorylation sites were predicted by two out of three prediction programs used (KinasePhos, PredPhospho and Group-based Phosphorylation Scoring).

 

Figure 2
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  		Fig. 2. Phylogenetic relationships between AmqKir channels and other metazoan inward-rectifier potassium (Kir) channels. The phylogenetic relationship was derived as described in Materials and methods. The best Maximum Likelihood tree is shown with Bayesian support values above and RaxML maximum likelihood bootstrap support values below each node. Relationships not recovered in the RaxML analysis are indicated by nf. The tree was simplified by collapsing vertebrate Kir subfamilies from one through to seven and urochordate, arthropod and nematode clades of inward-rectifiers (represented by open triangles). Complete datasets used in analysis are available upon request.

 

Figure 3
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  		Fig. 3. Heterologous expression of sponge inward-rectifier potassium (Kir) channels. Strongly rectifying currents in 5 mmol l–1 external K+ (KMES) recorded from (A) AmqKirA using 2 s voltage steps from –150 to +100 mV in +20 mV increments from a holding potential –50 mV and (B) AmqKirB using a voltage ramp from a holding potential of –50 mV. Current–voltage plots for AmqKirB taken at 10 (#1), 30 (#2), 60 (#3) and 90 (#4) mins. The inset shows, in the same cell, the time-dependent change in the normalized current at –140 mV (until the recording ended). (C) Effect of increasing external K+ on AmqKirA currents. Solutions of 2, 5, 10, 25 and 50 mmol l–1 KMES were used. The inset is a semi-log plot of the reversal potential versus external K+ concentration for AmqKirA (N=6–28 per concentration, means ± s.e.m.). The fitted line has a slope of 49 mV. (D) Representative AmqKirA current–voltage relationships recorded in 5 mmol l–1 K+, 100 mmol l–1 NMDG, and 100 mmol l–1 Na (all used as MES salts) showing no Na or NMDG permeability. The inset shows results of the test of Na (0.03–1 mmol l–1 NaCl) block of 5 mmol l–1 KMES currents (N=5, means ± s.e.m.).

 

Figure 4
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  		Fig. 4. Test of channel block of AmqKirA. Representative current–voltage relationships in 5 mmol l–1 KMES with increasing concentrations of (A) CsCl or (B) BaCl2. (C) The normalized Kir current at –140 mV is plotted versus blocking ion concentration. The data were fit by a logistic equation with an IC50 of 37 µmol l–1 for Ba2+ (N=5–8) and 173 µmol l–1 for Cs+ (N=8, means ± s.e.m.). Test for tertiapin-Q (TPN-Q) toxin block of (D) AmqKirA, (E) mKir2.1 (mouse Kir) and (F) rKir1.1b (rat Kir) in the absence (0 nmol l–1) or presence of 10 and 100 nmol l–1 TPN-Q. Recordings were done in 5 mmol l–1 KMES, pH 7.6. Currents were measured using 500 ms voltage ramps from a holding potential of –50 mV. The block of rKir1.1b was reversible.

 

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© The Company of Biologists Ltd 2009