A novel inwardly rectifying K+ channel, Kir2.5, is upregulated under chronic cold stress in fish cardiac myocytes

A new member of the inward-rectifier K(+) channel subfamily Kir2 was isolated and characterised from the crucian carp (Carassius carassius) heart. When expressed in COS-1 cells this 422 amino acid protein produced an inward-rectifying channel with distinct single-channel conductance, mean open time and open probability. Phylogenetic sequence comparisons indicate that it is not homologous to any known vertebrate Kir channel, yet belongs to the Kir2 subfamily. This novel crucian carp channel increases the number of vertebrate Kir2 channels to five, and has therefore been designated as ccKir2.5 (cc for Carassius carassius). In addition to the ccKir2.5 channel, the ccKir2.2 and ccKir2.1 channels were expressed in the crucian carp heart, ccKir2.1 being present only in trace amounts (<0.8% of all Kir2 transcripts). Whole-cell patch clamp in COS-1 cells demonstrated that ccKir2.5 is a stronger rectifier than ccKir2.2 or ccKir2.1, and therefore passes weakly outward current. Single-channel conductance, mean open time and open probability of ccKir2.5 were, respectively, 1.6, 4.96 and 4.17 times as large as that of ccKir2.2. ccKir2.5 was abundantly expressed in atrium and ventricle of the heart and in skeletal muscle, but was a minor component of Kir2 in brain, liver, gill and kidney. Noticeably, ccKir2.5 was strongly responsive to chronic cold exposure. In fish reared at 4 degrees C for 4 weeks, ccKir2.5 mRNA formed 59.1+/-2.1% and 65.6+/-3.2% of all ccKir2 transcripts in atrium and ventricle, respectively, while in fish maintained at 18 degrees C the corresponding transcript levels were only 16.2+/-1.7% and 23.3+/-1.7%. The increased expression of ccKir2.5 at 4 degrees C occurred at the expense of ccKir2.2, which was the main Kir2 isoform in 18 degrees C acclimated fish. A cold-induced increase in the slope conductance of the ventricular I K1 from 707+/-49 to 1001+/-59 pS pF(-1) (P<0.05) was thus associated with an isoform shift from ccKir2.2 towards ccKir2.5, suggesting that ccKir2.5 is a cold-adapted and ccKir2.2 a warm-adapted isoform of the inward-rectifying K+ channel.