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Kir7.1 Disease Mutation in the Inner Pore is Critical for K+ Permeation
Author(s) -
Beverley Katie Marie,
Steffen Jack Thomas,
Heyrman Joseph Andrew,
Shahi Pawan Kumar,
Pattnaik Bikash Ranjan
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04731
Subject(s) - fusion protein , microbiology and biotechnology , transport protein , transmembrane domain , wild type , green fluorescent protein , leucine zipper , point mutation , chemistry , biology , potassium channel , mutant , biophysics , amino acid , biochemistry , peptide sequence , gene , recombinant dna
Background Inwardly rectifying potassium (K+) channels (Kir) are critical for maintaining membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene that encodes for Kir7.1 protein, located in the retinal pigmented epithelium (RPE), contribute to the development of pediatric blindness. One such point mutation in the KCNJ13 gene c.458C>T changes Threonine (T) to Isoleucine (I) at amino acid position 153. T153 is located within the second transmembrane domain, lining the inner pore of the tetrameric protein. Hypothesis We hypothesized that T153I would not affect protein translation, assembly, or trafficking. Because of the specific location of T153 within the pore, the mutation might affect ion permeation through the channel. Methods HEK293 cells in culture were transfected with either GFP tagged wildtype (WT) Kir7.1 or T153I plasmid DNA. Protein from these cells was isolated and used for Western blotting and Mass‐spectroscopy. Confocal live‐cell imaging, using Nikon‐C2, mapped the expression of GFP fusion protein. Hoescht marked the nucleus, and WGA‐Alexa 594 labeled the plasma membrane. Images were subjected to off‐line analysis with NIS Elements. Whole‐cell patch‐clamp electrophysiology provided insight into channel function. For ion permeation, we exploited K+ and Rubidium (Rb+) permeability of the channel and analyzed using the Clampfit program. Results The T153I mutant protein size was identical to Kir7.1 WT protein. The specific protein band confirmed the Kir7.1 amino acid sequence. Live‐cell imaging indicated that both Kir7.1 and T153I 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 −943.49 ± 180.56 pA (n=6), but the T153I shows negligible inward current of −81.21 ± 20.29 pA (n=9, P=0.0051). Kir7.1 WT zero‐current potential was −62.21 ± 1.66 mV (n=6), however, the average T153I zero‐current potential was −5.98 ± 1.62 mV (n=9, P=6.2x10 −12 ). Rb+ ion, known to permeate readily through Kir7.1 channel, showed current fold‐change of 4.95 ± 0.78 (n=6) in WT, while T153I showed current fold‐change of 2.60 ± 0.50 (n=9, P=0.033). P‐value <0.05 is regarded as significant. Conclusion These data indicate that the T153I mutant channel is translated and trafficked to the membrane. However, our data indicate that T153I did not maintain WT zero‐current potential and decreased inward current compared to Kir7.1 WT. Surprisingly, T153I showed Rb+ permeability, albeit less compared to the WT channel. Since T153I is within the inner pore‐lining of the channel, it appears to constrict the channel to make it non‐permeable to K+, but Rb+ can pass through, resulting in a dysfunctional channel. Support or Funding Information University of Wisconsin‐Madison Endocrinology and Reproductive Physiology NICHD T32, NIH EY024995, NIH P30EY016665, NIH S10OD018221