IKCa Channels Are a Critical Determinant of the Slow AHP in CA1 Pyramidal Neurons

8/18/2019 IKCa Channels Are a Critical Determinant of the Slow AHP in CA1 Pyramidal Neurons

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Report

IKCa Channels Are a Critical Determinant of the Slow AHP in CA1 Pyramidal Neurons

Graphical Abstract

Highlights

d CA1 pyramidal cells express intermediate-conductance

Ca2+-activated K + channels

d An sAHP exhibits a pharmacological profile specific to IKCa

channels

d IKCa channels reduce temporal summation of EPSPs and

mediate spike accommodation

d IKCa channels are a key determinant of the sAHP in CA1

pyramidal cells

Authors

Brian King, Arsalan P. Rizwan, ...,

Gerald W. Zamponi, Ray W. Turner

Correspondence

[email protected]

In Brief

The molecular identity of a Ca2+-

dependent slow afterhyperpolarization

(sAHP) that controls cortical neuronal

excitability has gone unsolved for over 30

years. King et al. now show that IKCa

(KCa3.1) channels underlie the sAHP in

CA1 pyramidal cells to suppress temporal

summation of EPSPs and mediate spike

accommodation.

King et al., 2015, Cell Reports 11, 175–182 April 14, 2015 ª2015 The Authors

http://dx.doi.org/10.1016/j.celrep.2015.03.026

mailto:[email protected]://dx.doi.org/10.1016/j.celrep.2015.03.026http://crossmark.crossref.org/dialog/?doi=10.1016/j.celrep.2015.03.026&domain=pdfhttp://dx.doi.org/10.1016/j.celrep.2015.03.026mailto:[email protected]


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Cell Reports

Report

IKCa Channels Are a Critical Determinantof the Slow AHP in CA1 Pyramidal Neurons

Brian King,1,2 Arsalan P. Rizwan,1,2 Hadhimulya Asmara,1 Norman C. Heath,1 Jordan D.T. Engbers,1 Steven Dykstra,1

Theodore M. Bartoletti,1 Shahid Hameed,1 Gerald W. Zamponi,1 and Ray W. Turner 1,*1Hotchkiss Brain Institute, University of Calgary, Calgary, AB T2N 4N1, Canada2Co-first author

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.celrep.2015.03.026

This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

SUMMARY

Control over the frequency and pattern of neuronal

spike discharge depends on Ca

2+

-gated K

+

channelsthat reduce cell excitability by hyperpolarizing

the membrane potential. The Ca2+-dependent slow

afterhyperpolarization (sAHP) is one of the most

prominent inhibitory responses in the brain, with

sAHP amplitude linked to a host of circuit and

behavioral functions, yet the channel that underlies

the sAHP has defied identification for decades.

Here, we show that intermediate-conductance

Ca2+-dependent K + (IKCa) channels underlie the

sAHP generated by trains of synaptic input or post-

synaptic stimuli in CA1 hippocampal pyramidal cells.

These findings are significant in providing a molecu-

lar identity for the sAHP of central neurons that willidentify pharmacological tools capable of potentially

modifying the several behavioral or disease states

associated with the sAHP.

INTRODUCTION

Of the many classes of K+ channels recognized in central neu-

rons, few have as key a role in regulating the frequency and

pattern of spike discharge as Ca2+-gated K+ channels. Despite

this, only two classes of Ca2+-gated K+ channels were recog-

nized to control signal processing and spike output in central

neurons: big-conductance (BK) and small-conductance (SK)channels ( Adelman et al., 2012; Berkefeld et al., 2010 ). The prop-

erties of BK and SK channels differ in key respects that mediate

repolarization over relatively short time frames of activity, with

BK channels typically generating a fast afterhyperpolarization

of 10 ms and SK channels a medium afterhyperpolarization

of 100 ms. However, other Ca2+-gated K+ channels exert

even greater control over membrane excitability than BK or SK

channels. One of the longest-standing examples of this is the

Ca2+-gated slow afterhyperpolarization (sAHP) of seconds dura-

tion ( Andrade et al., 2012 ). The sAHP is unique in being highly

amenable to modulation by neurotransmitters, with sAHP ampli-

tude linked to circuit functions that include synaptic plasticity,

electroencephalography, aging, and several disease states ( Dis-

terhoft et al., 2004; Haug and Storm, 2000; Madison and Nicoll,

1986; Martı́n et al., 2001; Zhang et al., 2013 ).

Despite the wealth of information on sAHP properties, the mo-

lecular identity of the Ca2+-dependent K+ channel(s) underlying

the sAHP has defied explanation. A third possible Ca2+-depen-

dent channel is the ‘‘intermediate-conductance Ca2+-gated K+

channel’’ (SK4, IKCa, KCa3.1) ( Ishii et al., 1997; Joiner et al.,

1997; Logsdon et al., 1997 ). While these were not originally

thought to be expressed in CNS neurons (reviewed in Wulff

et al., 2007 ), recent work in cerebellar Purkinje cells and on

IKCa protein distribution suggests a wide potential expression

pattern in central neurons ( Engbers et al., 2012; Turner et al.,

2015 ). The current study tested the hypothesis that IKCa chan-

nels contribute to the Ca2+-dependent component of the sAHP

in CA1 pyramidal cells. We report that K+ channels of intermedi-

ate conductance with the unique pharmacological profile of IKCa

channels indeed underlie the sAHP, identifying the molecular ba-sis for one of the largest inhibitory responses in central neurons.

RESULTS

The sAHP of CA1 Pyramidal Cells

The sAHP in CA1 hippocampal pyramidal cells is known to

mediate spike accommodation, in which spike frequency is pro-

gressively reduced during an injected current pulse and followed

by a post-stimulus afterhyperpolarization ( Madison and Nicoll,

1986 ). The sAHP can also be evoked by synaptic inputs in stra-

tum radiatum (SR) using short stimulus bursts (5–30 pulses,

50 Hz), with the sAHP apparent during and immediately after

the stimulus train ( Figures 1 and 2 ). To test the functional roleof IKCa channels, we first blocked SK channels using 100 nM

apamin and Kv7 channels with 10 mM XE-991, given a role for

Kv7 channels in other hippocampal neurons ( Tzingounis et al.,

2010 ). It is known that IKCa channels are apamin insensitive

( Wulff et al., 2007 ), and we confirmed that 10 mM XE-991 had

no effect on IKCa channels ( Figure S1 A) and did not impede

SR-evoked synaptic transmission ( Vervaeke et al., 2006 ). Any

contribution from the Na-K pump was minimized by recording

at 32C ( Gulledge et al., 2013 ). To focus on excitatory synaptic

potentials, we applied 50 mM picrotoxin to block GABAergic

transmission. The combination of apamin, XE-991, and picro-

toxin produced little qualitative change in the firing response to

Cell Reports 11, 175–182, April 14, 2015 ª2015 The Authors 175

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current pulse injection or SR stimulus trains (see the Supple-

mental Experimental Procedures and Figures S2 A and S2B).

The role of IKCa channels can be distinguished using the se-

lective blockers TRAM-34, Senicapoc, or NS-6180 ( Stocker

et al., 2003; Strøbæk et al., 2013; Wulff et al., 2001 ). The selec-

tivity of TRAM-34 has been thoroughly established, with IKCa

channels exhibiting far greater sensitivity to 1 mM TRAM-34

than SK or BK channels ( Wulff et al., 2000 ). Control tests

confirmed that 1 mM TRAM-34 had no significant effect on BK,

Kv7.3, or TMEM16B channels expressed in tsA-201 cells or

the SR-evoked excitatory postsynaptic current paired-pulse ra-

tio in tissue slices ( Figures S1B–S1E). Since TRAM-34 must firstbe internalized to block IKCa channels, we found that the fastest

and most reliable block was obtained through internal perfusion

of 1 mM TRAM-34 through the electrode (see the Experimental

Procedures ).

IKCa Channels Contribute to Spike Accommodation and

Temporal Summation

Internal perfusion of 1 mM TRAM-34 reduced spike accommoda-

tion and the sAHP in rat CA1 pyramidal cells evoked by current

injection or a 30-pulse SR stimulus train ( Figures 1 A and 1B).

The initial suppression of excitatory postsynaptic potential

(EPSP) summation by the sAHP during a 50-Hz SR stimulus train

C

5

mV

5

mV

200 ms

TRAM-34

SR Stimulation, 30 pulses, 50 Hz

GABAergic inhibition intact

BA

TRAM-34

1st 500 ms last sec

500 1000 500 1000 F o l d F r e q u e n c y

F o l d N u m b e r

F o l d F r e q u e n c y

F o l d N u m b e r

20

mV

500 ms

024

6

8

0

2

4

Time frame (ms)

10

mV100 ms 1st 200 ms last 400 ms

1

2

(7)

(7)

00

2

4

6

200 400 200 400

10

mV

Time frame (ms)

SR Stimulation, 30 pulses, 50 Hz

10

mV

200 ms

** * * *

50 ms

200 ms

Figure 1. TRAM-34-Sensitive Mechanisms

Contribute to Spike Accommodation and

the sAHP

(A) Spike accommodation during depolarizing

current injection is reduced by 1 mM TRAM-34 to

increase spike number and frequency. Mean barplotsindicate thefold change in spike frequencyor

spike number over the times indicated by hori-

zontal bars following TRAM-34.

(B) Repetitive SR stimulation (30 pulses, 50 Hz) in

thesamecellas in (A)is associated with prominent

EPSPsuppression and spike accommodationthat

is reduced by infusion of 1 mM TRAM-34. Open

arrows and inset illustrate the effects of TRAM-34

on thesAHP that follows a synaptic train. Meanbar

plotsindicate thefold change in spike frequencyor

spike number over the times indicated by hori-

zontal bars following TRAM-34.

(C) Repetitive SR stimulation (30 pulses, 50 Hz) at

sub- or supra-threshold intensity in the absence of

picrotoxin to preserve inhibitory inputs reveals a

significant effect by TRAM-34 (1 mM) on EPSP

summation and spike output.

All recordings in (A) and (B) were obtained in

100 nM apamin, 10 mM XE-991, and 50 mM

picrotoxin, while those in (C) did not include

picrotoxin. In all cases, TRAM-34 was internally

perfused through the electrode. Values are mean

± SEM; * p < 0.05. See also Figures S1–S3.

could be extensive, with the membrane

potential often approaching or even fall-

ingbelow therestingpotential ( Figure1B).

Block of the sAHP by TRAM-34 led to

a maintained depolarization during SR-

evoked synaptic trains and a loss of spike

accommodation evident in an increase in the frequency and

number of spikes ( Figures 1 A and 1B). The magnitude of a

post-stimulus TRAM-34-sensitive sAHP was more prevalent

for SR-evoked trains compared to just threshold current-evoked

spike discharge ( Figures 1 A and 1B). This is expected, given a

known relation between sAHP amplitude and spike frequency

and number ( Madison and Nicoll, 1984 ), with threshold current

injection evoking spike output at 12.7 ± 2.3 Hz (n = 7) compared

to 36.1 ± 5.3 Hz for 50-Hz SR stimuli. Each of these results was

obtained in rats of age postnatal day 18 (P18) to P23 (n = 8) or

P90 (n = 2) and in mice ranging from P25 to P50 (n = 8) or P60

(n = 2). We further delivered SR stimulus trains when picrotoxinwasexcluded(n = 3) to determineif theeffectsof TRAM-34 could

be detected when inhibitory GABAergic inputs were intact.

These tests confirmed that TRAM-34-sensitive currents are

effective when GABAergic inputs are intact by modulating

temporal summation and spike accommodation for either sub-

or supra-threshold SR stimulation ( Figure 1C).

The Synaptically Evoked sAHP Is Reduced in KCa3.1 /

Mice

We next examined the ability to record the sAHP in KCa3.1 /

mice compared to wild-type (WT). We found that in response

to depolarizing current injection, pyramidal cells in KCa3.1 /

176 Cell Reports 11, 175–182, April 14, 2015 ª2015 The Authors


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MTx reduced outward current by 40% ± 7.6% (n = 5, p < 0.01)

and reversed at 94.1 ± 6.7 mV (n = 5) ( Figure 3C). Finally, inter-

nal perfusion of the electrodewith the catalytic subunit of protein

kinase A (PKA cat , 100 U/ml) reduced outward current by 35% ±

4.3% in five out of six cells (p < 0.001) that reversed at 97.6 ±

4. 7m V( n= 5) ( Figure3D).A comparison of these results revealed

that the degree of block by each of the compounds (TRAM-34,

ChTx, MTx, and PKA cat ) was not significantly different (one-

way ANOVA, p > 0.05). The results again reveal a K+ current in

CA1 pyramidal cells with the unique pharmacological profile of

IKCa channels.

CA1 Pyramidal Cells Exhibit Ca2+-Dependent

Intermediate Conductance K + Channels

We next determined if Ca2+-activated K+ channels of intermedi-

ate conductance could be recorded in pyramidal cells. For this,

we obtained somatic on-cell patch recordings using a HEPES-

buffered aCSF-based electrode solution to preserve intracellular

contents and native Ca2+ buffering mechanisms. IKCa current

was isolated using a set of blockers of voltage- or Ca 2+-gated

ion channels in both the electrode and external aCSF (see the

Supplemental Experimental Procedures and Table S1 ). Calcium

channels were not blocked in order to promote Ca2+ conduc-

tance acrossthe patch by delivering a setof spike-like depolariz-

ing commands through the electrode (50 Hz, 20 pulses, 5 ms,

80 mV). The membrane potential was subsequently stepped to

a set of steady-state potentials from

60 to +30mV with respectto the resting state to examine evoked currents. When recording

under conditions of 3.25 mM [K]o in the on-cell electrode, we

assumed that the resting potential under the patch approached

the value obtained in a separate set of perforated-patch record-

ings of 64 ± 1.5 mV (n = 12). To simplify interpretation, outward

current is presented with respect to the cell interior as an upward

deflection in all on-cell recordings.

We found that the train of spike-like commands was followed

by activation of non-inactivating single channels (n = 4) or even

macroscopic current (n = 6) ( Figures 4 A and 4B). Single-channel

amplitude revealed a mean conductance of 30.4 ± 5.8 pS (n = 4)

( Figure 4 A), a value within the range for IKCa channels reported

in other cell types ( Wulff et al., 2007 ). Both single channels

(n = 4) and macroscopic currents (n = 6) were rapidly reduced

by bath perfusion of 1 mM TRAM-34, which will cross the cell

membrane in regions outside of the on-cell patch ( Figures 4 A

and 4B). The I-V plot for the TRAM-34-sensitive currents evoked

by the spike-like stimulus train reversed 10 mV more negative

than the native resting potential ( Figure 4 A) and could exhibit

slight inward rectification at more hyperpolarized potentials

(n = 4/6).

To determine the ion selectivity of macroscopic currents, we

obtained on-cell recordings using an electrode solution contain-

ing 140 mM KCl to establish equimolar [K] across the membrane

patch, thus setting EK to 0 mV. By comparison, ECl under these

conditions was predicted to rest at 90 mV across the patch.

All other blockers in the electrode and bath are described in

the Supplemental Experimental Procedures. To apply the series

of spike-like depolarizing commands over a range that would

promote Ca2+ influx through the patch, the holding potential

was set to 65 mV and the membrane subsequently stepped

to potentials over a range of 40 mV to +120 mV ( D mV) with

respect to 65 mV. As before, we recorded non-inactivating

macroscopic currents that were reduced by 1 mM TRAM-34

(n = 6) ( Figure 4B). Importantly, the TRAM-34-sensitive currents

were confirmed as representing a K+ conductance by reversing

through 0 mV (EK ) on the I-V plot ( Figure 4B).

The Ca2+ sensitivity of these currents was established when

0.1 mM DC-EBIO increased peak macrocurrent in six out of nine cells ( Figure 4C). Subsequent perfusion of the membrane

permeable BAPTA-AM (10 mM) fully blocked evoked currents

(n = 3) ( Figure 4C). PKA-mediated phosphorylation is well known

to block the sAHP of CA1 pyramidal cells ( Lancaster et al., 1991;

Pedarzani et al., 1998 ) as well as IKCa channels ( Wong and

Schlichter, 2014 ). We found that 8-bromo-cyclic AMP (100 mM)

rapidly reduced channel activity or macroscopic outward current

in on-cell recordings (n = 5) ( Figure 4D). These data reveal that a

Ca2+-dependent intermediate-conductance K+ channel can be

recorded that is sensitive to TRAM-34, an internal Ca2+ chelator,

or elevation of cyclic AMP, all properties consistent with IKCa

channels.

Control

50

pA100 ms

Control

20

pA100 ms

-80 -60 -40 -20 20 40

10

30

mV

60

Ap

20

-10

10

20

30

40

50

-80 -60 -40 -20 20 40 60

A

B

Control

50

pA100 ms

-80 -60 -40 -20 20 40 60-20

20

40

60

80

Control

50

pA100 ms

-110 mV

60 mV

Subtraction

-5

10

20

30

-80 -60 -40 -20 20 40 60

C

D

Subtraction

-110 mV

60 mV

TRAM-34 (n = 7)

PKAcat (n = 5)ChTx (n = 7)

Subtraction

Subtraction

MTx (n = 5)

mV

Ap

mV

Ap

mV

Ap

Figure 3. CA1 Pyramidal Cells Exhibit an

Outward Current with a Pharmacological

Profile Consistent with IKCa Channels

Shown are outside-out recordings from pyramidal

cell somata with 1 mM [Ca]i in response to a

500-ms rampcommand from110 mVto +60 mV.Mean I-V plots reflect currents blocked by the

indicated agents following subtraction of test

fromcontrol recordings. IKCa current was isolated

using blockers described in the Supplemental

Experimental Procedures.

(A–D) In each case, outward current is voltage in-

dependent and reduced by (A) TRAM-34 (1 mM),

(B) ChTx(100 nM),(C)MTx(100nM), or(D)PKA cat

(100 U/ml). ChTx and MTx were focally pressure

ejected, while TRAM-34 and PKA cat were inter-

nally perfused in the patch electrode.

SEMs for mean values in I-V plots are indicated by

the shaded area.



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Synaptic Activation of IKCa Channels and the sAHP

To test for synaptic activation of IKCa channels in relation to the

sAHP, we again used on-cell recordings and short trains of

supra-threshold SR stimuli (five pulses, 50 Hz). We furtherapplied a 60-mV steady-state holding potential above the resting

membrane potential to the patch to increase the driving force for

K+ current, and we applied the same blockers as for single chan-

nels above ( Figure 4 ).

In 5 of27 cases, on-cell recordings yielded singlechannels that

were activated during or by the end of a five-pulse SR stimulus

train( Figure 5 A). A high rate of opening was apparent immediately

following the train followed by a persistent flickering state for

several seconds ( Figure 5 A). Alternatively, a macroscopic out-

ward current was recorded that had a rapid onset following the

SR stimulus train and remained open for a variable duration of

2–5s before spontaneously terminating (n = 5) ( Figure5B). Calcu-

lating the ensembleaverageof SR-evokedmacroscopiccurrents

revealed an outward current that peaked immediately following

the SR stimulus train and then dissipated over 4–5 s ( Figure 5B).

Bath application of 1 mM TRAM-34 rapidly reduced or blockedSR-evoked single channels (n = 5) or macrocurrents (n = 5) ( Fig-

ures 5 A and 5B). These results are important in providing the first

evidence for synaptic activation of an identified K+ channel with

activity that correlates to the sAHP of CA1 pyramidal cells.

The Ca2+-activated outward current underlying sAHP current

(I sAHP ) is most often recorded under voltage clamp following a

depolarizing step command of 1 s. We directly compared the

sensitivity of the step-evoked and SR-evoked I sAHP to TRAM-

34 application. Perforated-patch whole-cell recordings were

used to preserve intracellular Ca2+ buffering and outward cur-

rents evoked by five SR stimuli (50 Hz) or by a step command

to +60 mV (500 ms) ( Figures 5C and 5D). An outward current

A

∆ mV Control

TRAM-34

C

O2

O1

O2O3

O1C

-60

-40

-20

+20

2

pA

200 msec

+30

+30

0

(-65)

∆ 30 mV

RMP

∆ 80 mV

∆ -60 mV

[K] o = 3.25 mM

C u r r e n t ( p A )


Applied voltage f rom rest (∆ mV)

20RMP-20-40-60

-1.5

-1.0

-0.5

RMP

0.5

1.0

1.5

γ = 30 pS

C

Control

BAPTA-AM

DC-EBIO

50

pA1 sec

∆ -60 mV

∆ 30 mV

∆ 30 mV

RMP

∆ 80 mV

∆ -60 mV

-60

∆ mV

+30

-60

+30

D

Control

-60

∆ mV

5

pA1 sec∆ 30 mV

RMP

∆ 80 mV

∆ -60 mV

+30

-60

+30 8-bromo-cAMP

B∆ mV

n = 6-8

-6

-4

-2

0

2

4

6


-100 -80 -60 -40 -20 20 40 600

∆ 120 mV

-65 mV

∆ 80 mV

∆ -40 mV

ECl EK

TRAM-34

Subtraction

Control

5pA

2 sec

[K] o = 140 mM

+120

+120

-40

-40


Predicted transmembrane

potential (mV)

Figure 4. IKCa Channels Are Expressed in CA1 Pyramidal Cells

Shown are on-cell somatic recordings using a HEPES-buffered aCSF internal solution, with Ca2+

influx evoked by a repetitive spike-like command (50 Hz,

20 pulses, 5 ms, 80 mV) followed by steps to different steady-state potentials. The resting membrane potential (RMP) across the patch is presumed to be

approximately

65 mV for records in (A), (C), and (D) with 3.25 mM [K]o, while that for 140 mM [K]o in (B) was set at

65 mV. Voltage commands reflect the stepapplied to the electrode and displayed with net depolarizing commands upward. Current is displayed with respect to the cell interior with outward current as an

upward deflection. The mean I-V plot in (B) reflects TRAM-34-sensitive currents calculated by subtraction of test from control records.

(A)On-cell recordingusing 3.25 mM[K]oin theelectrode reveals single channel activityfollowing thepulse train that israpidlyreducedor blocked by1mM TRAM-

34. Current reversed approximately 10 mV from the resting state.

(B) On-cell recordings of macroscopic current using equimolar [K]o exhibits reversal of TRAM-34-sensitive current through 0 mV (EK ).

(C) On-cell recorded macroscopic current is enhanced by the IKCa agonist DC-EBIO (0.1 mM) and blocked by 10 mM BAPTA-AM.

(D) On-cell macroscopic current is reduced by 100 mM 8-bromo-cyclic AMP. Records in (B) show every second record for clarity.

The electrode and bath perfusate in all cases containedblockers as described in theSupplemental Experimental Procedures. Traces were filtered at400–500 Hz

(8-pole Bessel). Values are mean ± SEM.

Cell Reports 11, 175–182, April 14, 2015 ª2015 The Authors 179


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was recorded following the SR stimulus train in seven outof eight

cells with an amplitude of 47 ± 5 pA and duration of 6.8 ± 1.5 s

(n = 7). In the same cells a step command to +60 mV evoked

an I sAHP of 28 ± 11 pA and 6.3 ± 1.2 s (n = 7). Perfusion of

1 mM TRAM-34 reduced both the SR- and step-evoked outward

current (n = 7, p < 0.01) ( Figure 5C). If IKCa channels underlie

I sAHP , then the outward current should also be blocked by

ChTx. We thus repeated these tests in the additional presence

of 5 mM TEA to first block BK channels and found that pressure

ejection of 100 nM ChTx reduced the I sAHP evoked by either SR

stimulation (n = 6, p < 0.05) or a step command (n = 7, p < 0.05)( Figures 5C and 5D).

DISCUSSION

The current study reveals that IKCa channels contribute sub-

stantially to the Ca2+-dependent sAHP in CA1 hippocampal py-

ramidal cells. Support for this was obtained through recordings

of intermediate-conductance K+ channels evoked in relation to

the sAHP with the unique pharmacological profile for IKCa chan-

nels. These data are thus important in resolving the molecular

basis for one of the largest Ca2+-dependent inhibitory postsyn-

aptic responses in central neurons.

Ionic Contributions to the sAHP

Studies on the ionic basis for the sAHP of CA1 pyramidal cells

have considered several channel subtypes. Fluctuation analysis

implicated a K+ channel of 2–5 pS ( Sah and Isaacson, 1995 ).

The SK1 isoform was thus considered to underlie the sAHP,

but this was later discounted ( Bond et al., 2004 ). A role for

TMEM16B channels in mediating various aspects of repolariza-

tion has been proposed for CA1 cells using the drugs NFA and

NPPB ( Huang et al., 2012 ), but these drugs also block IKCa

channels ( Fioretti et al., 2004; Wulff et al., 2007 ). We further

found no block of TMEM16B channels by TRAM-34 when ex-pressed in tsA-201 cells, and the current isolated here reversed

through EK and not ECl. A role for voltage-gated Kv7 channels

(IM ) in generating an sAHP has been reported, but these are

less involved in CA1 cells ( Tzingounis et al., 2010 ) and we

consistently recorded a TRAM-34-sensitive sAHP in XE-991.

While sodium-dependent K+ channels can contribute to an

sAHP in other cells ( Zhang et al., 2010 ), the sAHP and I sAHP

recorded here is Ca2+ dependent ( Figures S4 A–S4C). A role

for the Na-K pump was proposed based on the effects of

ouabain at 35C using high-frequency current pulses ( Gulledge

et al., 2013 ). We found that the m ajority of the sAHP evoked by

this protocol at 35C was blocked by internal infusion of 1 mM

5

pA

1 sec

SR stimulation, 5 pulses, 50 Hz

n = 9

A

c

o

c

o

C

D

2

pA100 msec

SR stimulation, 5 pulses, 50 Hz

Control TRAM-342

pA200 msec

Control

TRAM-3410pA

1 sec

Control

TRAM-3420

pA1 secSR

-65 mV

-65 mV

60 mV

5

pA1 sec

Control

ChTx

-65 mV

60 mV

10

pA1 sec

SR stimulation

+60 mV

Control

ChTx

4

pA1 sec

SR

-65 mV

B

Control TRAM-34

Figure 5. IKCa Channels Are Evoked in CA1

Pyramidal Cells by Synaptic Stimulation

(A and B) On-cell recordings with a HEPES-buff-

ered aCSF electrode solution (3.25 mM [K]). Cur-

rents are illustrated with respect to the cell interior

(outward current upward). Currents were evokedusing a 50 Hz, five-pulse SR stimulus train with a

net 60 mV depolarized holding potential to in-

crease driving force for K+

across the patch. The

electrode and bath perfusate contained blockers

as described in the Supplemental Experimental

Procedures. (A) A single channel is activated dur-

ing and followinga five-pulse SR stimulustrain and

is blocked by bath perfusion of 1 mM TRAM-34.

Dashed lines depict open (o) and closed (c) states,

with the same example expanded below. (B) On-

cellrecordings of an outward macroscopic current

that opens for prolonged but variable periods of

time following SR stimulus trains (arrows) and is

reduced or blocked by TRAM-34. An ensemble

average from 9 SR stimulus trains (lowest trace)

reveals an average time course equivalent to

an sAHP. Transients in (B) reflect capacitive

transients from spontaneous spike discharge in

the cell.

(C and D) Comparison of outward current evoked

by SR stimulation to the I sAHP evoked by a step

command in perforated-patch recordings in the

presence of 100 nM apamin, 10 mM XE-991, and

50 mM picrotoxin (C and D), with 5 mM TEA further

included in (D) to block BK channels for tests with

ChTx. Currents were evoked by five supra-

threshold SR stimuli (50 Hz) or a 500-ms step to

60mV toevokeI sAHP andbathapply 1mM TRAM-

34 or pressure eject 100 nM ChTx. Results in (C)

and (D) are from separate cells.

Tracesin (A)and (B) were filtered at500 Hz andthe

expanded trace in (A) at 1 kHz (8-pole Bessel).

Values are mean ± SEM.



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TRAM-34, with a small remaining TRAM-34-insensitive compo-

nent subsequently blocked by 20 mM ouabain ( Figures S4D

and S4E).

Interestingly, Ca2+-dependent single channels of 19 pS

conductance were earlier reported in both tissue slices ( Limaand Marrion, 2007 ) and cultured neurons ( Lancaster et al.,

1991 ). However, in neither case was the molecular identity of

these channels attained. The sAHP in cultured hippocampal

neurons was further reduced by clotrimazole ( Shah et al.,

2001 ), a drug related to TRAM-34 but later recognized as

non-specific. The sAHP and spike accommodation was also

reported to be insensitive to ChTx ( Lancaster and Nicoll,

1987; Shah and Haylett, 2000 ). By comparison, we found

ChTx-sensitive current in outside-out and perforated-patch re-

cordings. The reason for this difference is unknown but might

reflect differences in internal Ca2+ buffering. Indeed, our most

stable recordings of IKCa channels and I sAHP were obtained

using on-cell or perforated-patch recordings that minimize a

disruption of [Ca]i.

IKCa Channels and the sAHP

Our current interpretation for the ionic basis of the sAHP in CA1

pyramidal cells is that the majority of the large-amplitude, Ca2+-

dependent early phase (4–5 s) of the sAHP is mediated by IKCa

channels.An additional smaller contribution canbe made by Kv7

channels or by the Na-K pump, although the Na-K pump is ex-

pected to be most active following intense activation. Delin-

eating IKCa channels as a key contributor to the sAHP in a

cortical pyramidal neuron is significant in that IKCa channels

were believed to be restricted to endothelial cells and activated

microglia in central regions ( Wulff et al., 2007 ). The reasons why

early probes for in situ hybridization did not detect KCa3.1 signal

in brain is unknown, since a signal from at least endothelial

KCa3.1 would be expected. However, recent work has shown

that IKCa channels can be recorded in cerebellar Purkinje cells

( Engbers et al., 2012 ) and GFP expression driven by the

KCa3.1 promoter suggested an even wider expression pattern

of IKCa ( Turner et al., 2015 ). Indeed, numerous central neurons

express an sAHP that shares many properties with the sAHP in

CA1 pyramidal cells, including Ca2+ dependence, insensitivity

to BK or SK channel blockers, and rapid block by kinase path-

ways, with a very close parallel found in enteric and myenteric

neurons ( Neylon et al., 2004; Vogalis et al., 2002 ). The finding

that IKCa-mediated suppression of temporal summation is still

active under conditions of intact GABAergic inhibition further en-

sures that the influence of IKCa channels will be present underphysiological conditions. We thus expect the role for IKCa chan-

nels in generating an sAHP to be widely applicable to other clas-

ses of central neurons.

EXPERIMENTAL PROCEDURES

Animal Care and Slice Preparation

Experiments wereconducted on P18–P24 maleSprague-Dawley rats(Charles

River) raised from timed-pregnant dams or on breeding colonies of P25–P50

C57BL/6 WT mice or P25–P60 KCa3.1 / mice (UC Davis) (as described

in Turner et al., 2015 ) according to approved procedures by the Canadian

Council of Animal Care. Details on in vitro slice preparation are provided in

the Supplemental Experimental Procedures.

Electrophysiology

A suite of patch-clamp recording techniques was used to record from rat or

mouse hippocampal CA1 pyramidal cells under current- or voltage-clamp

conditions in the in vitro slice preparation (3234C) and from tsA-201 cells

(22C). Details on recording equipment and patch recording configurations

can be found in the Supplemental Experimental Procedures.

Solutions and Drug Applications

The lipophilic drugs TRAM-34, Senicapoc, and NS-6180 must be internalized

to target an internal binding site ( Stocker et al., 2003; Strøbæk et al., 2013;

Wulff et al., 2001 ). Using bath perfusion of these drugs, channel activity was

reduced in on-cell recordings in 10–15 min while whole-cell recordings could

take 20–30 minto achieve full block. Themostreliable blockwas obtainedwith

TRAM-34 and Senicapoc, with infusion of 1 mM TRAM-34 by exchanging the

electrodesolution (ALAScientific Instruments)achieving a block of IKCachan-

nelsin


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Disterhoft, J.F., Wu, W.W., and Ohno,M. (2004). Biophysicalalterationsof hip-

pocampal pyramidal neurons in learning, ageing and Alzheimer’s disease.

Ageing Res. Rev. 3, 383–406.

Engbers, J.D.T., Anderson, D., Asmara, H., Rehak, R., Mehaffey, W.H.,

Hameed, S., McKay, B.E., Kruskic, M., Zamponi, G.W., and Turner, R.W.

(2012). Intermediate conductance calcium-activated potassium channels

modulate summation of parallel fiber input in cerebellar Purkinje cells. Proc.

Natl. Acad. Sci. USA 109, 2601–2606.

Fioretti, B., Castigli, E., Calzuola, I., Harper, A.A., Franciolini, F., and Catacuz-

zeno, L. (2004). NPPB block of the intermediate-conductance Ca2+-activated

K+ channel. Eur. J. Pharmacol. 497 , 1–6.

Gu, N., Vervaeke, K., and Storm, J.F. (2007). BK potassium channels facilitate

high-frequency firing and cause early spike frequency adaptation in rat CA1

hippocampal pyramidal cells. J. Physiol. 580, 859–882.

Gulledge,A.T., Dasari,S., Onoue, K., Stephens, E.K., Hasse, J.M., and Avesar,

D. (2013). A sodium-pump-mediated afterhyperpolarization in pyramidal neu-

rons. J. Neurosci. 33, 13025–13041.

Haug, T., and Storm, J.F. (2000). Protein kinase A mediates the modulation of

the slow Ca(2+)-dependent K(+) current, I(sAHP), by the neuropeptides CRF,

VIP, and CGRP in hippocampal pyramidal neurons. J. Neurophysiol. 83,

2071–2079.

Huang, W.C., Xiao, S., Huang, F., Harfe, B.D., Jan, Y.N., and Jan, L.Y. (2012).

Calcium-activated chloride channels (CaCCs) regulate action potential and

synaptic response in hippocampal neurons. Neuron 74, 179–192.

Ishii, T.M., Silvia, C., Hirschberg, B., Bond, C.T., Adelman, J.P., and Maylie, J.

(1997). A human intermediate conductance calcium-activated potassium

channel. Proc. Natl. Acad. Sci. USA 94, 11651–11656.

Joiner, W.J., Wang, L.Y., Tang, M.D., and Kaczmarek, L.K. (1997). hSK4, a

member of a novel subfamily of calcium-activated potassium channels.

Proc. Natl. Acad. Sci. USA 94, 11013–11018.

Lancaster, B., and Nicoll, R.A. (1987). Properties of two calcium-activated

hyperpolarizations in rat hippocampal neurones. J. Physiol. 389, 187–203.

Lancaster, B.,Nicoll,R.A., and Perkel, D.J. (1991). Calciumactivates twotypes

of potassium channels in rat hippocampal neurons in culture. J. Neurosci. 11,

23–30.

Lima, P.A., and Marrion, N.V. (2007). Mechanisms underlying activation of the

slow AHP in rat hippocampal neurons. Brain Res. 1150, 74–82.

Logsdon, N.J., Kang, J., Togo, J.A., Christian, E.P., and Aiyar, J. (1997).

A novel gene, hKCa4, encodes the calcium-activated potassium channel in

human T lymphocytes. J. Biol. Chem. 272, 32723–32726.

Madison,D.V., and Nicoll, R.A.(1984). Control of the repetitive dischargeof rat

CA 1 pyramidal neurones in vitro. J. Physiol. 354, 319–331.

Madison,D.V., and Nicoll, R.A.(1986). Cyclic adenosine 30,50-monophosphate

mediates beta-receptoractionsof noradrenalinein rat hippocampal pyramidal

cells. J. Physiol. 372, 245–259.

Martı́ n, E.D., Araque, A., and Bun ˜ o, W. (2001). Synaptic regulation of the slow

Ca2+-activated K+ current in hippocampal CA1 pyramidal neurons: implica-

tion in epileptogenesis. J. Neurophysiol. 86, 2878–2886.

Neylon, C.B., Nurgali, K., Hunne, B., Robbins,H.L., Moore, S., Chen, M.X., and

Furness, J.B. (2004). Intermediate-conductance calcium-activated potassiumchannels in enteric neurones of the mouse: pharmacological, molecular and

immunochemical evidence for their role in mediating the slow afterhyperpola-

rization. J. Neurochem. 90, 1414–1422.

Pedarzani,P., Krause,M., Haug,T., Storm, J.F., and Stühmer, W. (1998). Mod-

ulation of the Ca2+-activated K+ current sIAHP by a phosphatase-kinase bal-

ance under basal conditions in rat CA1 pyramidal neurons. J. Neurophysiol.

79, 3252–3256.

Sah,P., and Isaacson, J.S.(1995).Channelsunderlying theslow afterhyperpo-

larization in hippocampal pyramidal neurons: neurotransmitters modulate the

open probability. Neuron 15, 435–441.

Shah, M., and Haylett, D.G. (2000). Ca(2+) channels involved in the generation

of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neu-

rons. J. Neurophysiol. 83, 2554–2561.

Shah, M.M., Miscony, Z., Javadzadeh-Tabatabaie, M., Ganellin, C.R., and

Haylett, D.G. (2001). Clotrimazole analogues: effective blockers of the slow

afterhyperpolarization in cultured rat hippocampal pyramidal neurones. Br.

J. Pharmacol. 132, 889–898.

Stocker, J.W., De Franceschi, L., McNaughton-Smith, G.A., Corrocher, R.,

Beuzard, Y., and Brugnara, C. (2003). ICA-17043, a novel Gardos channel

blocker, prevents sickled red blood cell dehydration in vitro and in vivo in

SAD mice. Blood 101, 2412–2418.

Strøbæk, D., Brown, D.T., Jenkins, D.P., Chen, Y.J., Coleman, N., Ando, Y.,

Chiu, P., Jørgensen, S., Demnitz, J., Wulff, H., and Christophersen, P.

(2013). NS6180, a new K(Ca) 3.1 channel inhibitor prevents T-cell activation

andinflammation in a ratmodel of inflammatorybowel disease.Br. J.Pharma-

col. 168, 432–444.

Turner, R.W., Kruskic, M., Teves, M., Scheidl-Yee, T., Hameed, S., and Zam-

poni, G.W. (2015). Neuronal expression of the intermediate conductance cal-

cium-activated potassium channel KCa3.1 in the mammalian central nervous

system. Pflugers Arch. 467 , 311–328.

Tzingounis, A.V., Heidenreich, M., Kharkovets,T., Spitzmaul, G., Jensen, H.S.,

Nicoll, R.A., and Jentsch, T.J. (2010). The KCNQ5 potassium channel medi-

ates a component of the afterhyperpolarization current in mouse hippocam-

pus. Proc. Natl. Acad. Sci. USA 107 , 10232–10237.

Vervaeke, K., Gu, N.,Agdestein, C.,Hu, H., and Storm, J.F.(2006). Kv7/KCNQ/

M-channels in ratglutamatergic hippocampal axons and their rolein regulation

of excitability and transmitter release. J. Physiol. 576, 235–256.

Vogalis, F., Harvey, J.R., and Furness, J.B. (2002). TEA- and apamin-

resistant K(Ca) channelsin guinea-pigmyenteric neurons: slowAHP channels.

J. Physiol. 538, 421–433.

Wong,R., and Schlichter, L.C. (2014). PKA reduces the rat and human KCa3.1current, CaM binding, and Ca2+ signaling, which requires Ser332/334 in the

CaM-binding C terminus. J. Neurosci. 34, 13371–13383.

Wulff, H., Miller, M.J., Hansel, W., Grissmer, S., Cahalan, M.D., and Chandy,

K.G. (2000). Design of a potent and selective inhibitor of the intermediate-

conductance Ca2+-activated K+ channel,IKCa1: a potentialimmunosuppres-

sant. Proc. Natl. Acad. Sci. USA 97 , 8151–8156.

Wulff, H., Gutman, G.A., Cahalan, M.D., and Chandy, K.G. (2001). Delineation

of the clotrimazole/TRAM-34 binding site on the intermediate conductance

calcium-activated potassium channel, IKCa1. J. Biol. Chem. 276, 32040–

32045.

Wulff, H., Kolski-Andreaco, A., Sankaranarayanan, A., Sabatier, J.M., and

Shakkottai, V. (2007). Modulators of small- and intermediate-conductance

calcium-activated potassium channels and their therapeutic indications.

Curr. Med. Chem. 14, 1437–1457.

Zhang, L., Kolaj, M., and Renaud, L.P. (2010). Ca2+-dependent and Na+-

dependentK+ conductancescontributeto a slowAHP in thalamicparaventric-

ularnucleus neurons: a novel target for orexin receptors. J. Neurophysiol. 104,

2052–2062.

Zhang, L., Ouyang, M., Ganellin, C.R., and Thomas, S.A. (2013). The slow

afterhyperpolarization: a target of b1-adrenergic signaling in hippocampus-

dependent memory retrieval. J. Neurosci. 33, 5006–5016.



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Cell Reports

Supplemental Information

IKCa Channels Are a Critical Determinant

of the Slow AHP in CA1 Pyramidal Neurons Brian King, Arsalan P. Rizwan, Hadhimulya Asmara, Norman C. Heath, Jordan D.T.

Engbers, Steven Dykstra, Theodore M. Bartoletti, Shahid Hameed, Gerald W. Zamponi,

and Ray W. Turner


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1

Figure S1, related to Figure 1. Pharmacological isolation of IKCa channels

Whole-cell recordings obtained with alpha subunits of the indicated proteins transiently expressed in tsA-

201 cells and recorded at room temperature.(A) The Kv7 channel blocker XE-991 (10 µM) has no significant effect on IKCa channels. IKCa was

coexpressed with calmodulin and currents recorded in 1 µM [Ca]i in response to a ramp command.

(B - D) TRAM-34 (1 µM) has no significant effect on BK channels (B), Kv7.3 channels (C), orTMEM16B calcium-gated chloride channels recorded with 100 nM [Ca]i (D). TMEM16B currents are

rapidly blocked by 300 µM niflumic acid (NFA) in (D).

(E) TRAM-34 (1 µM) does not significantly affect the mean SR-evoked EPSC paired pulse ratio (20 msinterstimulus interval).

Values are mean ± SEM with sample values shown in brackets.


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2

Figure S2, related to Figures 1 and 2. Background channel blockers have minimal effects on thesAHP and I sAHP (A) Perfusion of 100 nM apamin, 10 µM XE-991, and 50 µM picrotoxin to block SK and Kv7 channels

and GABAergic transmission slightly reduce but do not eliminate spike accommodation during current pulse injection.

(B) The early phase of the sAHP following a train of SR input is blocked ( arrow) and the sAHP is more

evident after perfusion of apamin, XE-991, and picrotoxin (inset ).

(C) The sAHP evoked by a train of SR stimuli is progressively reduced by block of NMDA receptors (DL-AP5, 25 µM) and AMPA receptors (DNQX, 10 µM).(D, E) Recordings of IsAHP evoked as a tail current under whole-cell voltage clamp using a depolarizing

step command (D) or following a SR stimulus train (E). Perfusion of apamin, XE-991, and picrotoxin

selectively reduces the early component of outward current consistent with a block of SK channels. Spikesand stimulus artefacts in (B, C, E) and currents during the command pulse in (D) are truncated.


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Figure S4, related to the Discussion. The majority of the sAHP is calcium-dependent and TRAM-34-

sensitive(A, B) Representative whole-cell recordings of IsAHP evoked by a step command to +60 mV at 34ºC is blocked by perfusion of low extracellular calcium (a), as is an inward tail current recorded in another cell

in the presence of internal 1 µM TRAM-34 (B).

(C) A perforated patch whole-cell recording at the soma records an IsAHP of 6 sec duration following a

step command to +60 mV at 22 ºC. The IsAHP is amplified upon generation of a calcium current duringthe step command, visible as an all-or-none unclamped calcium spike (inset ). Recordings in (B) were

conducted using a KMeSO4-based internal solution with 0.1 EGTA, ATP, GTP, creatine, and 1 µM

TRAM-34.


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5

(D, E) Representative whole-cell recording from a rat CA1 pyramidal cell applying a stimulus protocol

consisting of 1 nA, 2 ms current pulses delivered at 50 Hz for 3 sec to test the role for the Na-K pump ingenerating the sAHP at 35ºC (as in Gulledge et al. 2013). Internal infusion of TRAM-34 (1 µM) blocks a

large component of the sAHP. Subsequent addition of bath perfused 20 µM ouabain blocked the small

remaining TRAM-insensitive component of the sAHP. All recordings in (D, E) were conducted in the

presence of 100 nM apamin, 10 µM XE-991, and 50 µM picrotoxin.(E) Bar plot of mean sAHP area following the end of the current pulse train for records such as shown in

(D).Values are mean ± SEM. *, p < 0.05. Sample numbers for mean values are shown in brackets.


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Table S1, related to Figures 3, 4 and 5. Values of Kd/IC50/EC50 and the actual applied concentrations of

agonists and antagonists used.

Inhibitors Actions Kd IC50 /EC50 Applied

External

Applied

Internal

References

Tetrodotoxin Nav1.1-1.4,

1.6-1.7

1-24 nM 1 μM (Catterall et al., 2005a;

Zimmer, 2010)

Apamin SK1-3 2-12 nM 100 nM (Sah and Faber, 2002)

TEA BK

Kv1.1

Kv1.6

Kv3.x

80-330 µM

0.5 mM

1.7-7 mM

0.09-0.3 mM

5 mM See (Coetzee et al., 1999)for pharmacology of TEA

4-AP Kv1.x

Kv2.x

Kv3.x

Kv4.2

0.1-1.5 mM

0.5-4.5 mM

0.02-1.2mM

2-5 mM

5 mM 2 mM (Coetzee et al., 1999)

CsCl HCN1-4 0.16-0.20 mM 2 mM (Stieber et al., 2005)

TRAM-34 KCa3.1 20-25 nM 20 nM 1 µM 1 µM (Jenkins et al., 2013; Wulffet al., 2001; Wulff et al.,

2000)

Senicapoc KCa3.1 11 nM 100 nM (Maezawa et al., 2012)

ChTx KCa3.1

Kv1.2

Kv1.3

Kv1.6

10 nM

1.7-17 nM

0.5-2.6 nM

1 nM

100 nM (Van Renterghem et al.,

1995)

(Coetzee et al., 1999)

Maurotoxin KCa3.1

Kv1.2

1 nM

0.1 nM

100 nM (Castle et al., 2003)

Ni2+ Cav3.x 50-300 µM 300 µM (Lee et al., 1999)

Cd2+ Cav1.x

Cav2.3

2.14 µM

0.8 µM

30 µM (Hobai et al., 1997)

(Catterall et al., 2005b)8-bromo-cAMP PKA-I and-

II

0.015-

0.019 nM

100 µM (Schwede et al., 2000)

(Hoffman and Johnston,

1998)

PKACat KCa3.1CaM-

bindingdomain

100 U/ml (Wong and Schlichter, 2014)

XE-991 Kv7.x 2.2 µM 10 µM (Schwarz et al., 2006)

Activators

DC-EBIO 1 µM 0.1 µM (Wulff and Castle, 2010)

SKA-31 KCa3.1 260 nM 1 µM (Sankaranarayanan et al.,

2009)


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Supplemental Experimental Procedures:

Slice preparation:

Animals were anaesthetized by isoflurane inhalation and 270 µm dorsal hippocampal slices cut in ice-cold

solution (mM): 215 sucrose, 25 NaHCO3, 20 D-glucose, 2.5 KCl, 0.5 CaCl2, 1.25 NaH2PO4 and 3 MgCl2

bubbled with carbogen gas. The slices were incubated for 10-15 min (34°C) in artificial cerebrospinal fluid

(aCSF) composed of (mM): 125 NaCl, 3.25 KCl, 1.5 CaCl2, 1.5 MgCl2, 25 NaHCO3, and 25 D-glucose

bubbled with carbogen gas. Slices were stored at room temperature until recordings conducted at 32-34 ºC

as a submerged preparation.

Evoking the sAHP

As earlier reported (Wu et al., 2004), the amplitude of the evoked sAHP could differ between cells, with

up to 20% exhibiting little or no detectable sAHP. The current study focused on those cells exhibiting a

detectable sAHP. The combination of apamin, XE-991, and picrotoxin slightly reduced but did not block

spike accommodation evoked during current pulse injection (Figure S2A; n = 8). There was also little

qualitative change in the firing response to SR stimulus trains, although the early phase of the subsequent

AHP (SK-mediated) was reduced and the sAHP was slightly more prominent, likely due to an increase in

calcium influx in the absence of SK channels (Figure S2B; n = 8). The ability to detect both AMPA- and

NMDA-mediated components of the SR-evoked sAHP was also maintained in the presence of apamin, XE-

991, and picrotoxin (Figure S2C; n = 8). Recording the IsAHP following a step command or a burst of SR

stimuli established that these compounds reduced primarily an early component of outward current

consistent with an SK-mediated response (Figures S2D and S2E; n = 8). Recordings of IsAHP using

whole-cell or perforated patch configurations were stable over 30 min time (Figure S3, n = 7), revealing

minimal influence of washout or a change in access resistance that could account for the actions of applied

drugs.

Patch recordingsPatch recordings were made using Multiclamp 700B amplifiers and Digidata 1440A with DC-10 kHz

bandpass filter and pClamp software. Pipettes were constructed from 1.5 mm O.D. fiber-filled glass (A-M

Systems) with resistance of 4-8 MΩ. Series resistance was compensated with bridge balance circuitry for

current clamp recordings and during voltage clamp recordings by up to 80% compensation. Negative bias

current of < 200 pA was applied during current clamp recordings to maintain a subthreshold resting

potential at ~-65 mV. For whole-cell experiments, control recordings were made >5 min after break in to

promote full stabilization with the internal solution. During whole-cell recording, cells were rejected for

any drift in access resistance of > 20%. On-cell recordings were obtained with a 10 kHz cutoff filter and

processed offline by filtering at 240-500 Hz (Bessel 8-pole).

Solutions and drug applications

Chemicals were obtained from Sigma unless noted. The following drugs were used to block the identified

ion channels to isolate IKCa: BK (TEA, 5 mM; IbTx, 100 nM), SK (apamin, 100 nM), Kv7 (XE-991, 10

µM), Kv4.x (4-AP, 5 mM external, 2 mM internal), Kv1.x (TEA, 5 mM; ChTx, 100 nM; MTx, 100 nM),

sodium (TTX, 200 nM - 1 µM), HVA calcium (CdCl2, 30 µM), LVA calcium (NiCl2, 300 µM), HCN

(external CsCl, 2 mM). TRAM-34 (1 µM) was applied most often by internal perfusion of the electrode

and by bath perfusion when indicated. Synaptic responses were blocked by: GABA-A (picrotoxin, 50 µM),


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8

GABA-B (CGP55485, 1 µM, Tocris), NMDA (DL-AP5, 25 µM, Ascent Scientific), and AMPA/KA

(DNQX, 10 µM, Tocris Scientific). NiCl2, CdCl2, CsCl, TEA, 4-AP, and picrotoxin were prepared daily

from stock solutions. DNQX, DC-EBIO, and SKA-31 were first dissolved in dimethylsulfoxide (DMSO)

(final DMSO < 0.1%). Senicapoc was a gift of H. Wulff (UC Davis). Pressure ejection of toxins were

carried out in a HEPES (10)-buffered aCSF of (mM): 150 NaCl, 3.25 KCl, 1.5 CaCl 2, 1.5 MgCl2, 10

HEPES, 20 D-glucose, pH 7.3. Pressure electrodes included BSA (0.1%) to reduce drug binding, and food

coloring (1:100) to visualize the region of drug ejection.

Patch recording configurations and media

External solution for all patch configurations: External solution (aCSF) was composed of (mM): 125

NaCl, 3.25 KCl, 1.5 CaCl2, 1.5 MgCl2, 25 NaHCO3, and 25 D-glucose bubbled with carbogen. All slice

recordings were conducted at 32 -34 º C.

Whole-cell internal: Current-clamp whole-cell recording solution consisted of (mM): 130 KMeSO3, 0.1

EGTA, 10 HEPES, 7 NaCl, 0.3 MgCl2, pH 7.3 with KOH, providing an ECl of -75 mV and EK of -97 mV; .

5 di-tris-creatine phosphate , 2 Tris-ATP and 0.5 Na-GTP were added daily from frozen stocks to whole-

cell recording solutions.

Perforated-Patch internal: Gramicidin-perforated patch recordings solution contained (mM): 10 KCl, 135

K-Gluconate, 10 HEPES, 1 MgCl2, 75 µg/ml gramicidin prepared in DMSO (< 0.01% DMSO in the

internal).

Outside-out internal: Outside-out voltage clamp recordings to isolate IKCa currents used an electrode

solution of (mM): 140 KCl, 2.83 MgCl2, 10 HEPES, 5 EGTA, 2 4-AP, 4.25 CaCl2 (1 µM [Ca]i,

Maxchelator Ca/Mg/ATP/EGTA Calculator, 0.165 ionic strength), pH 7.3 (EK -99 mV); 5 di-tris-creatine

phosphate, 2 Tris-ATP and 0.5 Na-GTP were added daily from frozen stocks to outside-out internal

solution. The external solution further contained synaptic blockers and TTX, apamin, XE-991, TEA, 4-AP,and CsCl to block SK, BK, Na, Kv1, Kv4, Kv7, LVA and HVA Ca

2+ channels, and HCN channels (as

above, and detailed in Table S1).

On-cell internal: On-cell recording electrodes to isolate IKCa channels contained HEPES-buffered aCSF

with (mM): 150 NaCl, 3.25 KCl, 1.5 CaCl2, 1.5 MgCl2, 10 HEPES and 20 D-glucose, pH 7.3. The electrode

solution further contained synaptic blockers and TTX, apamin, XE-991, TEA, 4-AP, and CsCl to block SK,

BK, Na, Kv1, Kv4, Kv7 channels (as above, and detailed in Table S1). The external bath solution for on-

cell recordings contained the same drugs as the electrode solution but did not contain excitatory synaptic

blockers (DL-AP5 and DNQX) or TTX to allow for synaptic excitation.

tsA-201 cells

tsA-201 cells were maintained as previously described (Engbers et al., 2012; Rehak et al., 2013) and

transiently transfected with cDNA (5 µg/µl) of Kv7.3, BK, TMEM16B or IKCa and calmodulin together

with cDNA for GFP to identify cells successfully transfected. Currents were recorded at room temperature

(22 ºC) in aCSF consisting of (mM): 120 NaCl, 3 NaHCO3, 4.2 KCl, 1.2 KH2PO4, 1.5 MgCl2, 10 D-

Glucose, 10 HEPES and 1.5 CaCl2 , pH adjusted to 7.3 with NaOH. The following internal solutions were

used for voltage-clamp recordings in tsA-201 cells. For recording IKCa channels, electrodes were filled


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9

with (mM): 140 KCl, 5 EGTA, 5 HEPES, 2.83 MgCl2, 4.25 CaCl2 (1.1 µM free [Ca]i, Maxchelator

Ca/Mg/ATP/EGTA Calculator, http://maxchelator.stanford.edu/CaMgATPEGTA-TS.htm, 0.165 ionic

strength), pH adjusted to 7.3 with KOH. For recording BK currents, electrodes were filled with (mM): 140

KCl, 5 HEPES, 0.1 EGTA, 0.5 MgCl, 5 ATP, 1 GTP, pH adjusted to 7.3. For recording Kv7.3 currents,

electrodes were filled with (mM): 140 KCl, 0.1 EGTA, 2.5 MgCl2, 10 HEPES, pH adjusted to 7.3. For

recording TMEM16B currents, electrodes were filled with (mM): 15.22 CsCl, 124.78 CsMeSO3, 5 EGTA,

10 HEPES, 1.73 MgCl2, 1.68 CaCl2 (100 nM free [Ca]i, Maxchelator Ca/Mg/ATP/EGTA Calculator,http://maxchelator.stanford.edu/CaMgATPEGTA-TS.htm), with ECl = -45 mV, and pH adjusted to 7.3.

TMEM16B currents were activated by giving 200 ms voltage steps from a holding potential of -40 mV.

Stimulation

Synaptic input was evoked using a concentric bipolar electrode (Frederick Haer, CBCMX75(JL2))

positioned in the mid SR and driven by a stimulus isolation unit (Digitimer , 0.1-0.2 ms pulse width).

Data Analysis and Statistical Methods

sAHP areas were measured as the area under (in current clamp mode) or over (in voltage clamp mode)

baseline, defined as the mean voltage current or voltage level preceding the current or voltage protocol,from 200 ms after the protocol to 7 secs after the protocol. Amplitude of the sAHP was measured as the

voltage level 200 ms after the current or stimulus protocol. Mean single channel conductance was

calculated for hyperpolarizing potentials where channel amplitude was best delineated. Data were analyzed

using Clampfit 10 software and custom Matlab R2007B scripts, and statistical analysis in OriginPro 8.

Paired-sample Student t-tests were used unless otherwise indicated.


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Wulff, H., Gutman, G.A., Cahalan, M.D., and Chandy, K.G. (2001). Delineation of the clotrimazole/TRAM-34

binding site on the intermediate conductance calcium-activated potassium channel, IKCa1. J. Biol. Chem. 276 ,

32040-32045.

Wulff, H., Miller, M.J., Hansel, W., Grissmer, S., Cahalan, M.D., and Chandy, K.G. (2000). Design of a potent and

selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential

immunosuppressant. Proc. Natl. Acad. Sci. U S A 97 , 8151-8156.

Zimmer, T. (2010). Effects of tetrodotoxin on the mammalian cardiovascular system. Mar. Drugs 8 , 741-762.

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IKCa Channels Are a Critical Determinant of the Slow AHP in CA1 Pyramidal Neurons