PUBLICATION

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development

Authors

Jędrychowska, J., Vardanyan, V., Wieczor, M., Marciniak, A., Czub, J., Amini, R., Jain, R., Shen, H., Choi, H., Kuznicki, J., Korzh, V.

ID

ZDB-PUB-240317-11

Date

2024

Source

Developmental Biology 513: 50-62 (Journal)

Registered Authors

Jain, Ruchi, Jędrychowska, Justyna, Korzh, Vladimir, Kuznicki, Jacek, Shen, Hongyuan

Keywords

CRISPR-Cas9 site-specific mutagenesis, Gain-of-function, Kinocilia, Loss-of-function, Otolith, Silent α-subunit, Zebrafish

MeSH Terms

Zebrafish Proteins*/genetics
Zebrafish Proteins*/metabolism
Voltage-Dependent Anion Channels*/genetics
Voltage-Dependent Anion Channels*/metabolism
Animals
Zebrafish*/embryology
Zebrafish*/genetics
Mutation/genetics
Ear*/embryology

(all 9)

PubMed

38492873 Full text @ Dev. Biol.

Abstract

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2^-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2^-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

Genes / Markers

Figures

Show all Figures

Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
GFP	kcng4b^{waw304/waw304}; s356tTg (AB)	standard conditions	Protruding-mouth	inner ear hair cell	IFL	Fig. S7
GFP	kcng4b^{waw304/waw304}; s356tTg (AB)	standard conditions	Prim-5	inner ear hair cell	IFL	Fig. S7
GFP	kcng4b^{waw304/waw304}; s356tTg (AB)	standard conditions	Protruding-mouth	posterior lateral line neuromast hair cell	IFL	Fig. S7
GFP	kcng4b^{waw305/waw305}; s356tTg (AB)	standard conditions	Prim-5	inner ear hair cell	IFL	Fig. S8
GFP	kcng4b^{waw305/waw305}; s356tTg (AB)	standard conditions	Protruding-mouth	inner ear hair cell	IFL	Fig. S8
GFP	kcng4b^{waw305/waw305}; s356tTg (AB)	standard conditions	Protruding-mouth	posterior lateral line neuromast hair cell	IFL	Fig. S8
GFP	s356tTg (AB)	standard conditions	Protruding-mouth	inner ear hair cell	IFL	Fig. S7
GFP	s356tTg (AB)	standard conditions	Prim-5	inner ear hair cell	IFL	Fig. S7
GFP	s356tTg (AB)	standard conditions	Prim-5	inner ear hair cell	IFL	Fig. S8
GFP	s356tTg (AB)	standard conditions	Protruding-mouth	inner ear hair cell	IFL	Fig. S8

1 - 10 of 20

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
kcnb1^sq301/sq301 (AB)	standard conditions	Prim-15	otic vesicle increased size, abnormal	Fig. S1
kcnb1^sq301/sq301 (AB)	standard conditions	Prim-15	otolith increased amount, abnormal	Fig. S1
kcnb1^sq301/sq301 (AB)	standard conditions	Long-pec	otic vesicle increased size, abnormal	Fig. S1
kcnb1^sq301/sq301 (AB)	standard conditions	Long-pec	otolith increased amount, abnormal	Fig. S1
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	heart morphology, abnormal	Fig. S3
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	macula kinocilium normal amount, normal	Fig. 7
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	otic vesicle increased size, abnormal	Fig. 5
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	otolith increased amount, abnormal	Fig. 5
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	third ventricle cell delaminated, abnormal	Fig. S3
kcng4b^{waw304/waw304} (AB)	standard conditions	Prim-5	ventricular system hydrocephalic, abnormal	Fig. S3

1 - 10 of 31

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
s356tTg	Tg(pou4f3:GAP-GFP)	Transgenic Insertion
sq301		Indel	kcnb1
waw304		Small Deletion	kcng4b
waw305		Small Deletion	kcng4b

Human Disease / Model

Sequence Targeting Reagents

Fish

Fish
AB
kcnb1^sq301/sq301 (AB)
kcng4b^{waw304/waw304} (AB)
kcng4b^{waw304/waw304}; s356tTg (AB)
kcng4b^{waw305/waw305} (AB)
kcng4b^{waw305/waw305}; s356tTg (AB)
s356tTg (AB)

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
GFP	EFG	GFP

Mapping

Jędrychowska et al., 2024 ZDB-PUB-240317-11 PMID:38492873

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development

Jędrychowska et al., 2024

ZDB-PUB-240317-11
PMID:38492873