DOI: 10.5061/dryad.6hdr7sr8t
title: A novel autism-associated KCNB1 mutation dramatically slows Kv2.1 potassium channel gating activation, deactivation and inactivation

description: KCNB1, on human chromosome 20q13.3, encodes the alpha subunit of the Kv2.1
 voltage gated potassium channel. Kv2.1 is ubiquitously expressed
 throughout the brain and is critical in controlling neuronal excitability,
 including in the hippocampus and pyramidal neurons. Human KCNB1
 mutations are known to cause global development delay or plateauing,
 epilepsy, and behavioral disorders. Here, we report a sibling
 pair with developmental delay, absence seizures, autism spectrum disorder,
 hypotonia, and dysmorphic features. Whole exome sequencing
 revealed a heterozygous variant of uncertain significance (c. 342
 C&gt;A, p. (S114R) in KCNB1, encoding a serine to arginine
 substitution (S114R) in the N-terminal cytoplasmic region of Kv2.1. The
 siblings' father demonstrated autistic features and was
 determined to be an obligate KCNB1 c. 342 C&gt;A carrier based on
 familial genetic testing results. Functional investigation of
 Kv2.1-S114R using cellular electrophysiology revealed slowing of channel
 activation, deactivation, and inactivation, resulting in
 increased net current after longer membrane depolarizations. To our
 knowledge, this is the first study of its kind that compares the
 presentation of siblings each with a KCNB1 disorder. Our study
 demonstrates that Kv2.1-S114R has profound cellular and
 phenotypic consequences. Understanding the mechanisms underlying
 KCNB1-linked disorders aids clinicians in diagnosis and treatment
 and provides potential therapeutic avenues to pursue.
description: <strong>Human genome sequencing</strong><br>We
 received a signed case report consent form from the legal guardian of the
 children. Both siblings had whole exome sequencing performed at GeneDx
 (Gaithersburg, MD, USA) using paired-end reads on an Illumina platform.
 Sequence reads were aligned to human genome build GRcH37/USCS hg19. Data
 was filtered using GeneDx’s custom analysis tool (XomeAnalyzer). The
 variant was reported as a variant of uncertain significance in
 accordance with the American College of Medical Genetics and Genomics
 (ACMG) criteria based on transcript NM_004975.2 17.
 <strong>Preparation of channel subunit cRNA preparation and
 Xenopus laevis oocyte injection</strong><br>cDNA encoding
 human KCNB1 was sub-cloned into a Xenopus expression vector
 (pMAX) incorporating <em>Xenopus</em>
 <em>laevis</em> β-globin 5’ and 3’ UTRs flanking the coding
 region to enhance translation and cRNA stability by Genscript (Piscataway,
 NJ, USA). The mutant KCNB1 construct was generated by Genscript and
 subcloned into pMAX as above. cRNA transcripts were generated by in vitro
 transcription using the T7 mMessage mMachine kit (Thermo
 Fisher Scientific, Waltham, MA, USA) according to manufacturer’s
 instructions, after vector linearization with PacI. Stage V and VI
 defolliculated <em>Xenopus laevis</em> oocytes (Xenoocyte,
 Dexter, MI, USA) were injected with the channel cRNAs (2 ng) and incubated
 at 16 oC in Barth’s solution containing penicillin and streptomycin, with
 daily washing, prior to two-electrode voltage-clamp
 (TEVC) recording. <strong>Two-electrode voltage
 clamp (TEVC)</strong><br>TEVC was conducted at room
 temperature with an OC-725C amplifier (Warner Instruments, Hamden, CT,
 USA) and pClamp10 software (Molecular Devices, Sunnyvale, CA, USA)
 24 hours after cRNA injection. Oocytes, in a small-volume oocyte bath
 (Warner), were viewed with a dissection microscope for cellular
 electrophysiology. Extracellular bath solution (in mM): 96 NaCl, 4 KCl, 1
 MgCl2, 0.3 CaCl2, and 10 HEPES, adjusted to pH 7.6 with TRIS
 BASE. Solutions were introduced into the oocyte recording bath by gravity
 perfusion at a constant flow of 1 ml per minute. Pipettes (1-2 MΩ
 resistance) were filled with 3 M KCl. Current-voltage graphs were measured
 in response to voltage pulses between -80 mV and +40 mV at 10 mV intervals
 from a holding potential of -80 mV. Conductance graphs were measured
 from tail currents generated at -40 mV immediately following the prepulse
 and normalized to the maximal current. Conductance was plotted as a
 function of voltage and fitted with a single Boltzmann
 function.  <strong>Activation and Deactivation
 Kinetics</strong><br>Activation kinetics were measured in
 response to voltage pulses between -10 mV and + 40 mV at 10 mV intervals
 from a holding potential of -80 mV. Deactivation kinetics were
 measured between -120 mV and -60 mV in 10 mV intervals immediately
 following a +40 mV prepulse from a holding potential of -80 mV. Activation
 and deactivation traces were each fitted with a single exponential
 function. <strong>Inactivation, Fraction of
 non-inactivated channels, and Recovery from
 inactivation</strong><br>Inactivation was measured in response
 to a single voltage pulse at +40 mV for 10s and 20s from a holding
 potential of -80 mV. The percentage of inactivation was derived from the
 difference between the peak and plateau of the current at +40 mV. Fraction
 of non-inactivated channels was measured in response to 10s voltage pulses
 between -80 mV and +10 mV immediately prior to a +40 mV voltage pulse from
 a holding potential of -80 mV. Fraction of non-inactivated channels was
 measured from the +40 mV voltage pulse, normalized to the maximal current,
 and fitted with a single Boltzmann function (Eq. 1). Recovery from
 inactivation was measured in response to consecutive 5s +40 mV pulses at
 interpulse intervals of increasing duration from 0.01 to 30 seconds. The
 subsequent peaks of these pulses were then divided against the initial
 pulse and plotted as a function of the interpulse interval. 
 <strong>Statistics and
 Reproducibility</strong><br>At least 2 batches of oocytes were
 used per experiment. Multiple comparison statistics were conducted using a
 One-way ANOVA with a Dunnett's test. Comparison of two groups was
 conducted using a t-test; all p values were two-sided. All
 electrophysiological data and statistics are summarized in
 the Supplementary Tables and Statistics file. All data were analyzed using
 Clampfit (Molecular Devices) and Graphpad Prism software (GraphPad, San
 Diego, CA, USA), stating values as mean ± SEM.
description: # A novel autism-associated KCNB1 mutation dramatically slows Kv2.1
 potassium channel gating activation, deactivation and inactivation The
 datasets included are the original Excel files used to generate each panel
 for figures 2-4 in this manuscript. The title of each Excel file is
 labeled to directly correspond to the figure in the manuscript: Figure
 Number & panel &gt; Channel Investigated &gt; Parameter Measured The
 types of files included within this repository are as follows: ## IV These
 data were used to generate current-voltage relationships for WT Kv2.1,
 Kv2.1-S114R, and Kv2.1/Kv2.1-S144R. Data in these files were measured from
 the peak of the prepulse current. ## Gmax These data were used to generate
 conductance-voltage curves for WT Kv2.1, Kv2.1-S114R, and
 Kv2.1/Kv2.1-S144R. Graphs were generated by taking measurements from the
 tail current (-40 mV) immediately following the prepulse current. ## Erev
 These data were used to estimate the reversal potential for WT Kv2.1,
 Kv2.1-S114R, and Kv2.1/Kv2.1-S144R. ## Current density comparisons These
 data were taken from the values used to generate the IV curve from the
 prepulse currents of WT Kv2.1, Kv2.1-S114R, and Kv2.1/Kv2.1-S144R at +40
 mV. ## Activation and Deactivation These data were used to determine the
 time constants (Tau) of WT Kv2.1, Kv2.1-S114R, and Kv2.1/Kv2.1-S144R.
 Activation and deactivation curves were fitted using a single exponential.
 ## Inactivation These data were generated by holding WT Kv2.1,
 Kv2.1-S114R, or Kv2.1/Kv2.1-S144R for 10 or 20 seconds at +40 mV and
 measuring the percentage inactivation between the peak current at the
 beginning of pulse and the final current at the end of the pulse. ##
 Fraction of non-inactivated channels These data were generated by
 measuring Kv2.1, Kv2.1-S114R, or Kv2.1/Kv2.1-S144R currents at +40 mV
 immediately following a 10 second holding pulse from -80 to 10 mV in 10 mV
 increments. Current densities at +40 mV were normalized to maximal current
 and fitted with a single Boltzmann curve. ## Recovery from inactivation
 These data were generated by measuring Kv2.1, Kv2.1-S114R, or
 Kv2.1/Kv2.1-S144R peaks in response to consecutive pulses at increasing
 durations. Values were divided against an initial pulse and plotted as a
 function of time. All statistical analysis were conducted as either paired
 t-test or one-way ANOVA with Dunnett's correction for multiple
 comparison.