solving structural topology of romk1 final from 102313
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Solving the Unique Structural Topology
of the ROMK1 Channel and Revelations in
Potassium Channel Biology
Emergence of a New Structural Family of
Potassium Channels -- the Inward Rectifier
Potassium (Kir) Channel Family
Initial Hydrophobicity Plots of ROMK1
• Sequence comparison of the predicted ROMK1 protein against GenBank,
EMBL, SWISS-PROT databases revealed no similarities; direct comparison to
all known K+ channel proteins revealed no significant sequence similarities
• Kyte-Doolittle & Eisenberg hydropathy analyses identified two potential
transmembrane segments flanked by hydrophilic segments
Amino acid no.
Original figure from K. Ho lab notebook, April, 1992KHv102313
Slo Ca2+-activated K+ channel Kv channels
ROMK1 channel
Identifying the K+ Channel Hydrophobicity Signature
• Major insight: comparison of
hydropathy plots of known K+ channel
subfamilies yielded a distinct K+
channel hydropathy “signature”
• Consisting of pore-forming H5 region
flanked by two transmembrane
segments, S5 and S6, in Kv channels
and Ca2+-activated K+ channels and a
preceding S4 segmentOriginal figures from K. Ho lab notebook, April, 1992KHv102313
Creating the ROMK1 Topological Model
• ROMK1 shared the same K+
channel signature: H5, P regions
with flanking transmembrane
segments, M1 and M2, an
amphipathic M0 region, and N-
glycosylation site
• ROMK1 H5, P regions exhibited
similarities of 44%, 59%
compared to Shaker Kv regionsK. Ho original figures:
Laboratory notebook, April 20, 1992
University of Oxford symposium lecture, July 30, 1992
Kyte-Doolittle
Hydropathy &
Garnier prediction
algorithm plots
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ROMK1 Conserves Pore-Forming H5 & P Regions
Yellen G. Nature 2002;419:35-42
Original figure from K. Ho lab notebook, April, 1992
P Region
ROMK P segment
Welling PA, Ho K. Am J Physiol
297:F849-F863 (2009)KcsA Pore
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Predicting ROMK1 Pore Characteristics
• ROMK1 P region conserved the T(V/L/I)GYG (T141/I142/G143/Y144/G145)
motif required for K+ selectivity -- providing further validation of its role
• Ser130 + Arg147 predicted ROMK1 insensitivity to external TEA+
based on site-directed mutagenesis studies in Shaker Kv channels
• Val140 and Ile142 predicted altered NH4+ and Rb+/K+ permeability in comparison to the Shaker H5 region
Original pencil drawings from K. Ho lab notebook, April-May, 1992KHv102313
ROMK1 Defined a New K+ Channel Family, Kir
• While ROMK1 conserved a homologous pore-forming P-region, it
differed from all known K+ channels (superfamily of voltage-gated &
second messenger-gated channels) by lacking the canonical structure
of six transmembrane segments, as well as, a S4 segment
• Thus ROMK1 (Kir1.1a) represented the defining member of the two-
transmembrane family of K+ channels consisting of inward rectifiers
Original figure from K. Ho lab notebook, April, 1992
Hibino H et al. Physiol Rev 2010:90:291-366
Earliest ROMK1 Structural Model
MacKinnon R. Nobel lecture: Potassiumchannels and the atomic basis of selectiveion conduction. Biosci Rep 2004;24:75-100
P Segment
K+ Selectivity Filter
KcsA channel
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Inward Rectifier (Kir) K+ Channel Family
• Kir channels have diverse functions in
the control of membrane excitability,
neuronal signalling, heart rate, vascular
tone, insulin release, and electrolyte
transport across epithelia
• Seven subfamilies Kir1.0 to Kir7.0 are
characterized by differences in degree of
rectification and regulation by specific
cellular signals
Bichet D et al. Nature Rev Neurosci 2003;4:957-967
Rapedius M et al. EMBO Reports 2006;7:611-616
Hibino H et al. Physiol Rev 2010:90:291-366
Tetrameric
ROMK
Channel
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Identifying the ROMK1 M0 Region
• In place of a Kv channel S4
segment (voltage-sensor), ROMK1
had an amphipathic segment, M0,
with limited similarity
• Suggesting that the M0 segment:– accounted for ROMK1’s lack of
voltage-dependence
– interacted with the lipid bilayer given
its intermediate hydrophobicity K. Ho original figures:
Laboratory notebook, April 20, 1992
University of Oxford symposium lecture, July 30, 1992
Kyte-Doolittle
Hydropathy &
Garnier prediction
algorithm plots
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M0 Segment Corresponds to the Kir “Slide Helix”
• The “slide helix” has been proposed to transduce the force between binding of
intracellular modulators to the cytoplasmic N-terminus and the M1
transmembrane segment leading to a M2 conformational change contributing to
channel opening; lateral “sliding” opens the channel gate
Kuo A et al. Science 2003;300:1922-1926
Ho K et al. Nature 1993;362:31-38
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