An introduction to KCNQ2 mechanisms

and paths towards mechanism-based

therapy

Kristen Park, MD

Colorado Children’s

Univ. of Colorado Univ.

John Millichap, MD

Lurie Children’s

Northwestern Univ.

RIKEE collaborators

Ed Cooper, MD, PhD

Associate Professor

Neurology, Neuroscience,

Molecular & Human Genetics

BCM, Houston TX

NINDS

CURE,

Jack Pribaz

GSK

Disclosure

Grant from GSK for studies of Potiga combined

treatment with sodium channel blockers

Consultant for SciFluor, Inc.

To: Cooper, Edward CSubject: KCNQ2

Dr. Cooper,

My wife and I saw the article regarding your research on KCNQ and had to reach out to you. Our son was recently diagnosed with a mutation in the KCNQ2 gene. That diagnosis has given us new hope and focus. For xxxx we had no diagnosis after xxxx of tests. He experienced seizures his first day of life and is very developmentally delayed. The seizures are now under control for the most part but his development and muscle tone are our biggest concerns.

Our doctor says he is the only KCNQ2 patient they have. From all that we have heard, this is a very rare condition. We are eager to hear what you think and to get involved with your research anyway we can.

Please let me know how we can help one another, or anything else that we can do to get closer to a cure. Thank you for your time and we look forward to hearing your thoughts.

Xxxxxxxxxx xxxxxxxxx

Genetics reports are more confusing to

patients and to us than we acknowledge

A change from C to T at

one position in the gene

A change from A to V in

the corresponding

position of the KCNQ2

protein

“KCNQ2 c.881C>T; p.A294V”

What does this mean? What are the limits of

current understanding? Are there a set of

explanations that can we can share generally?

Key background

1. The brain runs on

electricity, but...

salt molecules (ions) are the signal carriers

(not electrons)

2. Ion channels - the brain’s signaling “switches”,

are built according to a common plan, but come

in a many different varieties (~400 genes)

3. K-CN-Q-2

Chemical symbol for potassium

Short for “channel”

Subfamily of potassium channels “Q” (KCNA, B, C....)

2nd member of the “Q subfamily of K channels”

Understanding “genes”

We have many

genes

Each cell uses a

subset of those

genes tailored to

its own function

Each gene’s “recipe”

is written in a long

string (of DNA)

Genome is like a

vast recipe collection

Different recipes for

breakfast, dinner,

seasons, special

occasion

That’s where the

numbers come from

The cell converts (reads) the DNA string to a

protein string

21 3 4 5 6 7 8 9 DNA position

3 “codon” position21

3 Protein position21

. . . ~2600 in KCNQ2

Amino acids

. . . ~872 in KCNQ2

Start

Proteins are the molecular machines for

most of the body’s functions

How can long strings be made into machines?

• Amino acids are different from each other:

– “Oily” vs. “watery”

– Positive vs. negative

– Big vs. small

Amino acids have different properties

+ - oily

Amino acids have different sizes....(and packing matters)

-

oily

The 3-dimensional shape (and

moving parts) of each protein

are built from the folded amino

acid chain

1

2

3

4

5

6 78

9

10

11

12

“anatomy of an ion channel” – e.g., KCNQ2!

......872 x 4 =

3488 beads

per KCNQ2

channel!!!

watery amino acids face

outside of cell

watery amino acids face

watery inside of cell

oily amino acids face

the cell membrane (it’s oily)

oily amino acids make

up the interior of the protein

(mostly)

1. It’s buried in the oily cell membrane, so

“anatomy of an ion channel”

......872

watery amino acids

watery amino acids

oily amino acids

oily amino acids

buried within the

protein

2. add a hole in the middle, filled with water (the ion pore)

pore facing amino acids are watery

“anatomy of an ion channel”

......872

outside

inside

3. add a “gate” (a “moving part”)

+closed

+open

sensor

gate

_

_

Why do some KCNQ2 variants cause mild,

but others cause severe disease?

Severe variants are “substitutions” that replace one

amino acid with another of different chemical properties.

The change results in a “packing problem” or a “folding

problem”

Due to location within the channel, some mutation of

this type can allow one subunit to poison the function of

up to 3 normal subunits, resulting in up to 16-fold

(92.5%), but not complete, loss of channel activity.

The first few encephalopathy mutations suggested

a mechanism: 3 functional “Achilles’ heels”

Weckhuysen... Berkovic , Scheffer, de Jonghe, 2012; Millichap and Cooper, 2012

pore domain

vsd 1 vsd 3

Normal channel function

185-214

234-322

pore domain

vsd 1 vsd 3

Variant type 1: Pore block

234-322

185-214

pore domain

vsd 1 vsd 3

Variant type 2: Voltage-sensor can’t move

234-322

185-214

Variant type 3: can’t reach cell surface

Millichap and Cooper 2012

Inside

surface

membrane

calmodulin

X

One copy of a missense mutation can lead to mild transient neonatal epilepsy (BFNS), and reduces channel activity – but only slightly

0.75

current, fraction ofcontrol

Schroeder (1998)

Nature 396, 687

0 0.5 1

wt Q3 + ½Q2

wt Q2 + wt Q3

Q3 +

½Q2 + ½Q2 mut

Chrom 20 Chrom 8

Q3Q2

How one copy of some KCNQ2 variants

cause more severe loss of activity

1 : 4 : 6 : 4 :1 1 : 2 : 1

Channels formed by 4

KCNQ2 subunits,

50% mutant

Channels formed by 2

KCNQ2 subunits, 50% mutant

and 2 KCNQ3 subunits

Basic studies provide a rationale for KCNQ

opener drug treatment (1)

1. Neurons use very little of the KCNQ channel’s maximal

capacity

1% maximal KCNQ

activity1 msec

Battefeld et al., 2014

Rationale for drug treatment (2)

2. when channel gates are maximally opened by strong

membrane depolarization (without drug treatment), they

are still closed most of the time

single channel recording Li and Shapiro Journal of Neuroscience

2004

Rationale for drug treatment (summary)

1. Without drugs, the maximal channel opening possible

experimentally is only 10% (KCNQ2) to 30% (KCNQ2/3).

2. Neuron can only open channel 3-10% of that maximum,

because nerve signals are short in duration.

3. Therefore, channel is only “used” at .3 to 3% of maximal

capacity

4. With a powerful enough drug, should be able to increase

activity considerably

5. 8 fold difference between worst possible suppression

and a mild, transient syndrome: BFNS.

Challenges to implementing drug treatment

1. Currently available drug (Potiga) is relatively

low potency, low selectivity for KCNQ2/3, has

known side effects. Max clinical dose is about

1/10 what is used in the lab

2. Introducing new drugs is difficult and

expensive, especially for children.

3. Diagnosis is often delayed.

Solutions: our energy, skills, and dedication to the

task...

Intro to KCNQ2: conclusions

1. KCNQ2 is a needed molecule for preventing

seizures and promoting early brain

development; severity of related illnesses

appears to relate to degree of deficiency

2. KCNQ2 deficiency is never complete and drug

therapy is a strategy to augment activity of the

remaining normal channels

3. A large number of KCNQ opener drugs are

known but clinical development so far is limited

4. Many questions and challenges remain for

current research

Acknowledgements

BCM/TCH

Mingxuan Xu, Bao Tran, Li Li, Zhigang Ji

RIKEE Network

Lionel Carment, Universite de Montreal Marc Patterson, Mayo

Eric Marsh, Xilma Ortiz-Gonzalez, Emily Robbins Children’s Hospital of Philadelphia Bruria Ben Ze’ev Tel Aviv Molly Tracy Hasbro Children’s, Brown Univ.

Tammy Tsuchida, Phil Pearl (now BCH), Children’s National (DC)

John Millichap, Lurie Children’s (Northwestern U.)

Kristen Park, Paul Levisohn, Colorado Children’s (Aurora, CO)

Brenda Porter, Packard Children’s (Stanford)

UPENN

Zongming Pan Steve Scherer Amy Brooks-Kayal

Tingching Kao Steve Cranstoun Yogi Raol

Other Collaborations

Van Bennett (Duke) Jurgen Schwarz (Hamburg) Hugh Bostock (UCL) Ryuji Kaji(Tokushima) Yasushi Okamura, Atsuo Nishino (NIPS) Maarten Kole (Amsterdam, NIN) David Brown (UCL), Mala Shah (UCL)

Funders: NINDS, Miles Family Fund, AES/EF, Jack Pribaz Foundation, CURE