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

Initial public presentation of proposed structural model for an inwardly rectifying potassium channel subunit (Kir 1.1, ROMK1) on July

30, 1992 by K. Ho. The Physiological Society 1992 Research Symposium on ATP-Regulated Potassium Channels, University of Oxford.