Side‐Chain Polarity of Amino Acids within the Kir7.1 Channel Pore Lining Determine Permeability and Function

Abstract

Background Inwardly rectifying potassium (K+) channels (Kir) maintain membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene encoding for Kir7.1 protein, in the retinal pigmented epithelium (RPE), cause pediatric blindness. One point mutation in the KCNJ13 gene c.458C>T Threonine (T) to Isoleucine (I) at amino acid position 153 lines the inner pore of the tetrameric protein. We sought to elucidate the impact of pore region amino-acid hydrophobicity and side-chain length on the channel function. Hypothesis Amino acid side-chain polarity and length at position T153 are important determinants of Kir7.1 channel structure/function. Methods HEK293 cells in culture were transfected with GFP tagged wildtype (WT) Kir7.1, T153I, T153G, T153A, T153L, T153C, or T153S plasmid DNA. Live-cell imaging, using the Nikon-C2 confocal system, mapped the expression of GFP fused protein. Hoescht and WGA-Alexa 594 labeled the nucleus and plasma membrane, respectively. Off-line image analysis was performed with Nikon NIS Elements. Whole-cell patch-clamp electrophysiology with either extracellular Ringer's or Rb+ determined channel function. Extracellular K+-gradient was used to determine chord conductance. Data analyzed using the Clampfit program. Results Live-cell imaging indicated that Kir7.1 WT and all mutants studied are trafficked to the membrane. The IV plot for the Kir7.1 WT showed inward current measured at -150 mV with a mean amplitude of -863.67 ± 142.44 pA (n = 8) compared to Ile -68.54 ± 10.49 pA (n = 9, P = 2.79x10-5). Extracellular K+ dependent chord conductance further confirmed that T153I is nonfunctional. The current amplitude rank order for the mutant channel K+ permeability was Cys > WT > Ser > Gly > Ile > Ala> Leu. The membrane potential, measured as zero-current potential, was -57.75 ± 3.23 mV (n = 8) for the WT compared to-5.46 ± 6.01 mV (n = 9, P = 2.27x10-6) for the disease mutant with a rank order of Cys > WT > Ser > Leu > Gly > Ile > Ala. Rb+ ion, selectively permeates through Kir7.1 channel with a Rb+-current fold-change of7.28 ± 1.63 (n = 8) for WT versus 6.48 ± 2.78 (n = 9, P=0.81) for the disease mutant. The rank order of Rb+-current fold-change was Leu > Ser > WT > Ile > Cys > Gly > Ala. Conclusion Even though all the mutant channels traffick normally to the membrane, T153I was non-functional as determined by K+ ion permeability. Upon testing a more permeable ion, Rb+, it was deemed that T153I is actually dysfunctional. Based on the rank order, a substitution of polar amino acid at position 153 is required for Kir7.1 channel function. Permeability of all the mutant channels except T153A to Rb+ indicates that channel function is dependent on size of the cation. Since aa position T153 is within the inner pore-lining of the channel, a hydrophobic aa might alter protein-lipid interaction to affect channel function. Additionally, short-chain amino acids may further alter protein-protein interactions within the channel.