Cloning of a cardiac inward rectifier k^+ channel and its gating mechanisms

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We have isolated a cDNA coding for an inward rectifier K^+ channel (RBHIK1) from rabbit heart. The cloned cDNA encodes a protein of 427 amino acids with two putative transmembrane segments. The primary structure of RBHIK1 is highly homologous to that of IRK1 which is an inward rectifier K^+ channel cloned from mouse macrophage by expression cloning. Howere, there are some amino acid sequence differences observed between RBHIK1 and IRK1 in the segment between the first transmembrane segment and putative pore forming region, which may give rise to some functional differences between RBHIK1 and IRK1. RNA blot analysis revealed the expression of RBHIK1 mRNA (approximate estimated size of 5.5 kb) in various rabbit tissues. There are remarkable differences of the expression of RBHIK1 mRNA between the atrium and ventricle. The highest expression of the mRNA was detected in the ventricle, but no expression was detected in the atrium. The expression pattern is in good agreement with electrophys … More iological findings about the different sizes of native inward rectifier K^+ current in the ventricle and atrium.2. THe RBHIK1 current showed strong inward rectification. The amplitude of RBHIK1 current, like native inward rectifier K^+ current, increased as extracellular K^+ concentration increased and the current was blocked by Ba^<2+> or Cs^+ in a voltage- and time-dependent manner. However, although RBHIK1 was cloned from rabbit heart, unlike native cardiac inward rectifier, it exhibited no substantial outward current and negative slope conductance when expressed in Xenopus cocyte. In patch clamp experiments with 145 mMK^+ in pipette at 20-22ﾟC,the single channel conductance of the RBHIK1 channel was 17.8(]SY.+-.])0.47 pS,and that of the native inward rectifier K^+ channel was 23.5(]SY.+-.])0.29 pS.The currents of subconductance state of 2/3 of the full conductance frequently occurred in recordings of the single RBHIK1 channel. The RBHIK1 channel may be composed of three tetrameric constructs. Less