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First published online June 13, 2008
Journal of Experimental Biology 211, 2162-2171 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016121
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A novel inwardly rectifying K+ channel, Kir2.5, is upregulated under chronic cold stress in fish cardiac myocytes

Minna Hassinen, Vesa Paajanen and Matti Vornanen*

Faculty of Biosciences, University of Joensuu, PO Box 111, 80101 Joensuu, Finland


Figure 1
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  		Fig. 1. Amino acid alignment of ccKir2.5 channel with other members of the vertebrate Kir2 subfamily (A) and sequence similarities of vertebrate Kir channels (B). (A) Functionally important transmembrane domains (M1 and M2) and the intervening pore domain (P) are indicated with horizontal lines. Amino acids involved in Ba2+, polyamine and phosphatidylinositol 4,5-bisphosphate binding are identified with filled circles, filled triangles and asterisks, respectively. (B) Nucleic acid identity (above the line) and amino acid identity (below the line) of vertebrate Kir2.1 (top) and Kir2.2 (middle). Species included are: crucian carp (cc; EU182582 for Kir2.1 and EU182583 for Kir2.2), rainbow trout (om; DQ435674 and DQ435676), human (h; NP_000882 and NP_066292), guinea-pig (gp; Q549A2 and AAG17048), rat (r; NP_058992 and NP_446433) and mouse (m; NP_032451 and NP_034733). Nucleotide and amino acid sequence identities and similarities of crucian carp Kir2.5 (EU182584) with all known mouse Kirs are shown at the bottom, including Kir1.1 (NM_001012387), Kir2.1 (NM_008425), Kir2.2 (NM_010603), Kir2.3 (NM_008427), Kir2.4 (NM_145963), Kir3.1 (NM_008426), Kir3.2 (NM_001025584), Kir3.3 (NM_008429), Kir3.4 (NM_010605), Kir4.1 (NM_001039484), Kir4.2 (NM_001039057), Kir5.1 (NM_010604), Kir6.1 (NM_008428), Kir6.2 (NM_010602) and Kir7.1 (XM_001473740). Zebrafish Kir6.3 (NM_001012387) is also included.

 

Figure 2
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  		Fig. 2. A rooted phylogenetic tree of the vertebrate Kir channel family based on the amino acid sequences. KirBac3.1 was used as an outgroup. Numbers at the nodes indicate the bootstrap values for the clade of 1000 replications. Arrowheads indicate positions of the crucian carp Kir2 proteins in the tree. Predicted inward-rectifier potassium channels of the zebrafish are indicated with an asterisk. GenBank accession numbers for the mouse (m) Kir sequences are the same as in Fig. 1B and those for zebrafish (z) are as follows: Kir1.1, NM_001045169; Kir2.1, XM_687101; Kir2.2, XM_692512; Kir2.5, XM_001335914; Kir3.2, XM_692892; Kir3.3, XM_695527; Kir4.1, XM_001342957; Kir6.1, NM_001030153; Kir6.2, NM_001039827; Kir6.3, NM_001012387; and Kir7.1, NM_001045549.

 

Figure 3
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  		Fig. 3. Expression levels of ccKir2.1, ccKir2.2 and ccKir2.5 mRNAs in different tissues of the cold-acclimated crucian carp. The amount of ccKir2 mRNAs was normalised to the DnaJA2 expression level. Values are means ± s.e.m. of three pooled samples.

 

Figure 4
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  		Fig. 4. Characterisation of the cloned ccKir2 channels in COS-1 cells and native IK1 of the crucian carp ventricular myocytes by Ba2+ sensitivity and inward rectification. (A) Representative whole-cell recordings of the inward-rectifying K+ current through the cloned ccKir2.5 channel and IK1 of the warm-acclimated (WA) crucian carp myocyte, demonstrating a reversible block by 0.3 mmol l–1 external Ba2+. On the basis of current density, expression levels of the three ccKir2 proteins were similar (P>0.8). (B) Dose–response relationship of Ba2+ block of the cloned ccKir2 proteins and endogenous IK1 of the ventricular myocytes. The upper panel shows dose–response curves and the lower panels give mean values of Kd (left) and Hill coefficient (H, right), respectively. CA, cold-acclimated carp. Different letters indicate statistically significant differences between the cloned ccKir2 channels. Number of tested cells is 7–11. (C) Current–voltage relationship of cloned ccKir2 channels (left) and IK1 of warm- and cold-acclimated ventricular myocytes (right). The lower panels show the relative outward current for the same recordings. Note the higher inward and outward current density of cold-acclimated myocytes in comparison to warm-acclimated myocytes. (D) Boltzmann fits (top) and half-voltage (V1/2) and effective valency (z) of the inward rectification (bottom). Different letters indicate a statistically significant difference between cloned ccKir2 channels and an asterisk indicates a statistically significant difference between acclimation groups. Number of tested cells is 7–13.

 

Figure 5
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  		Fig. 5. Single-channel characteristics of the cloned ccKir2 channels from COS-1 cells and endogenous Kir2 channels of the crucian carp ventricular myocytes. (A) Comparison of inside-out recordings of the cloned ccKir2 proteins (left) and cell-attached recordings of the endogenous IK1 currents (right) demonstrating three distinct channel types on the basis of kinetics and conductance. Horizontal lines indicate the zero current level. (B) Current–voltage relationships of the ccKir2.1, ccKir2.2 and ccKir2.5 proteins in inside-out recordings from COS-1 cells (left) and cell-attached recordings of the endogenous IK1 in ventricular myocytes (right). Endogenous currents are means of all inward-rectifier channel types present in ventricular myocytes. (C) Conductance variation of endogenous ccKir2 currents of crucian carp ventricular myocytes in cell-attached (top) and inside-out (bottom) recordings. Distinct populations of small conductance (10–14 pS) channels are indicated with arrows. (D) Comparison of open time distribution (left) and open probability (right) of the cloned ccKir2.5 and ccKir2.2 channels and the endogenous fast and slow IK1 of ventricular myocytes. Open time distributions of the endogenous channels of temperature-acclimated fish include all recordings of the particular current type (slow IK1 or fast IK1): good fits to a monoexponential function suggest the presence of two separate channel types with different kinetics. Note also the different open time distributions of the cloned ccKir2.2 and ccKi2.5 channels. (E) Comparison of heterologously expressed ccKir2.5 and ccKir2.2 channels and endogenous slow and fast IK1 on the basis of mean open time ({tau}o) and open probability (Po).

 

Figure 6
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  		Fig. 6. Transcript abundance of ccKir2.1, ccKir2.2 and ccKir2.5 in atrium and ventricle of cold- and warm-acclimated crucian carp. A statistically significant difference (P<0.05) between acclimation groups is indicated by an asterisk.

 

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© The Company of Biologists Ltd 2008