Solving Structural Topology of ROMK1 FINAL from 102313
Transcript of Solving Structural Topology of ROMK1 FINAL from 102313
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
KHv102313
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
KHv102313
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
KHv102313
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
KHv102313
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
KHv102313
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
KHv102313