Potassium channel expression in the larval Drosophila melanogaster CNS Kathryn Scheckel

School of Life Sciences, Arizona State University, Tempe, AZ 85287

Methods 4

Introduction and Results

Ionic currents underlie action potential firing, excitability and synaptic transmission in all neurons. Proper regulation of ionic currents through strict control of channel expression levels and correct targeting of specific ion channel proteins is essential for normal behavior in every living organism. An emergent theme during recent years has been that alterations in gene expression of some genes coding for ionic currents may result in atypical regulation of the expression of other ion channels in a compensatory manner, so that excitability in all neurons can remain at a stable level. This mechanism is known as homeostatic regulation of neural excitability. This study aimed to understand the genetic interplay of potassium channel expression on the mRNA level in Drosophila to reveal potential mechanisms behind such homeostatic regulation of neural excitability. We focused on voltage and calcium activated fast-activating/fast-inactivating potassium channels. In Drosophila, these are coded for by the genes: Slowpoke (abbrev. slo), Small Conductance Calcium-Activated K+ (SK), Shaker, Shal, and Ether-a-go-go (eag).

Results The relative abundance of different voltage and calcium activated potassium channels in the larval ventral nerve cord of Drosophila.

Pan-neuronal RNAi knockdown shows no significant change for most potassium channels under investigation.

Shal RNAi knockdown in particular does not decrease Shal mRNA levels when expressed pan-neuronally under the control of Elav-GAL4, despite electrophysiological data showing the knockdown is effective when expressed under the control of motoneuron- specific GAL4 drivers.

Acknowledgments Dr. Carsten Duch, Dr. Navdeep Mutti, Benjamin Strauber, Fernando Vonhoff, and The Barrett Honors College at Arizona State University.

1

2To examine the effects of RNAi for one potassium channel on the expression of multiple potassium channels, specific RNAi transgene constructs were expressed pan-neuronally with the Elav-GAL4 driver in Drosophila larvae (third-instar phase). Each potassium channel's mRNA was then measured in the larval CNS using quantitative real-time polymerase chain reaction (qRT-PCR) and interpreted as relative changes

using the - ÄÄCT2 method (Livak, 2001).

The quality of an RNAi knockdown can be tested on the mRNA level by qRT-PCR, and on the protein level by Western Blotting or immunohistochemistry. In this work, a specific Shal antibody was used (Tsunoda et al., 2009) to analyze Shal protein levels and localization following Shal RNAi knockdown. Primary Shal AB was applied following fixation, coupled to secondary Cy-5 conjugated AB, and preparations were visualized by confocal microscopy (for detail see Ryglewski, Duch, 2009).

Drosophila

Gene

Vertebrate

Homologue Resulting Current

slo BK large conductance calcium activated k current

SK SK small conductance calcium activated k current

Shaker Kv1 A-type potassium current

Shal Kv4 A-type potassium current

eag Kv11 can result in multiple currents

Potassium Channels in Drosophila and Corresponding Vertebrate Homologues

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4

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1. qRT-PCR reveals the relative expression levels of different potassium channel mRNAs. Surprisingly, voltage activated A-type channels are present at much lower levels than calcium activated channels. Potassium channels Shab and Shaw (Kv2 and Kv3 in vertebrates) remain to be tested. 2. Pan-neuronal RNAi knockdown for slo under the control of Elav-GAL4 reduces slo mRNA levels by 30%, but does not affect any other tested potassium channel mRNA levels. Therefore, the slo UAS-RNAi construct is a useful tool. 3. However, for all other potassium channels (Kv1, Kv4, Kv11, SK) no significant reductions in mRNA levels were observed following pan-neuronal RNAi expression. 4. This is not because the RNAi constructs have no effect, as the same RNAis expressed in motoneurons under the control of various different GAL4 drivers (C380, D42) cause a significant reduction in the respective currents as shown for Shal in the previous section.5. Therefore, Elav-GAL4 might not express at sufficient strength or sufficient duration during development to cause significant UAS-RNAi knockdown, even when including extra DICER as in the case of Shal DICER.6. Nevertheless, this data suggest functional interactions between slo and eag expression, and between Shal and SK expression. In fact, recent unpublished electrophysiological data suggest that eag might underlie IK . (Ca)

7. In summary, qRT-PCR is useful to determine pan-neuronal ion channel mRNA expression. However, although Elav-GAL4 is commonly used in many applications, it is not a useful tool for pan-neuronal ion channel RNAi knockdown. The use of RNAi constructs has to be carefully validated for each mRNA under investigation, and also in each expression system.

Why do most RNAi knockdowns not reduce their respective channel mRNAs in the CNS?

Conclusions

Shal RNAi yields Shal knockdown when expressed under the control of motoneuron-specific GAL4 drivers, but not when expressed under control of pan-neuronal driver Elav-GAL4

Shal RNAi in motoneuronsabolishes Shal current

Ryglewski S , Duch C J Neurophysiol 2009;102:3673-3688

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Possibility 1: qRT-PCR is not sensitive enough. Possibility 2: The Elav-GAL4 driver does not produce sufficient expression levels in the CNS, though it is commonly used as a pan-neuronal driver.Possibility 3: Potassium ion channel RNAi expression in neurons causes compensatory up-regulation of transcription. This seems unlikely, however, because levels for channel mRNAs remain unaltered. We also know from electrophysiological recordings following RNAi expression in motoneurons that this is not the case.

Shal DICER RNAi ventral nerve cord

control ventral nerve cord control neuromuscular junction

Shal DICER RNAi neuromuscular junction

ShalNc82 + Shal Shal Nc82 Nc82 + Shal

Nc82 + Shal Nc82 + ShalNc82ShalShal

Shal RNAi in all neurons does not abolish Shal protein

Endogenous mRNA expression levels of calcium and voltage activated potassium channels

Expression levels of potassium channel mRNA following pan-neuronal RNAi knockdown

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4

slo

y = 0.0045x + 5.1932

R2 = 0.8352

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2

4

6

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10

12

0 50 100 150 200

Concentration (ng)

Ct(

slo

)-C

t(tu

b)

SK

y = 0.0013x + 6.8259

R2 = 0.1171

0

2

4

6

8

10

12

0 50 100 150 200

Concentration (ng)

Ct(

sk)-

Ct(

tub)

Shaker

y = 0.0141x + 8.319

R2 = 0.6815

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2

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6

8

10

12

0 50 100 150 200

Concentration (ng)

Ct(

sh)-

Ct(

tub)

Shal

y = 0.006x + 7.1049

R² = 0.67118

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2

4

6

8

10

12

0 50 100 150 200

Concentration (ng)

Ct(

shal)

-Ct(

tub)

eag

y = 0.0034x + 10.083R2 = 0.8781

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10

12

0 50 100 150 200

Concentration (ng)

Ct(

eag)-

Ct(

tub)

mRNAs for calcium-activated potassium channels (slo and SK) are present at much higher levels than mRNAs for A-type channels. Within A-type channels, there is much more Shaker than Shal mRNA. Also, eag (Kv11) mRNA is present at very high levels, but it remains unclear what currents may result from eag channel expression.

slo RNAi causes 30% reduction in slo mRNA. 1By comparison, in a slo hypomorph mutant

, slo mRNA is reduced by 50%. Furthermore, slo RNAi does not affect mRNA levels of any other potassium channel, indicating no homeostatic compensation.

(Elkins et al., 1986)

§ slo RNAi seems to be a useful tool for a partial but slo-specific knockdown.§However, none of the other potassium channel RNAis yield channel specific knockdowns when expressed under the control of Elav-GAL4.§Still, in slo hypomorphs, eag mRNA levels are significantly decreased.§Shal RNAi expressed with Elav-GAL4 does not affect Shal mRNA levels but significantly reduces SK RNA levels.

Relative mRNA expression levels following 1slo RNAi and slo mutant knockdown

Primer efficacy is similar for all mRNAs under investigation, thus allowing for a relative comparison of potassium channel mRNA expression levels.

Relative potassium channel mRNA expression levels in the CNS

Nc82 (green); Shal (red)

Gene

0

0.1

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0.5

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0.7

0.8

slo SK Shaker Shal eag

Re

lativ

e Q

ua

ntif

ica

tion

control RNAi mutant

*

*

0

0.1

0.2

0.3

0.4

0.5

slo SK Shaker Shal eagGene

Rela

tive Q

uantifica

tion

Gene Control RNAiStatistical

Significance

slo 0.6459 0.4703 Trend (p = 0.10)

SK 0.4502 0.5257 N.S.

Shaker 0.1205 0.1444 N.S.

Shal 0.0193 0.0235 N.S.

eag 0.4396 0.4475 N.S.

slo 0.6459 0.2623 *

SK 0.4502 0.4630 N.S.

Shaker 0.1205 0.1378 N.S.

Shal 0.0193 0.0238 N.S.

eag 0.4396 0.1552 *

slo 0.5333 0.5267 N.S.

SK 0.5525 0.4864 N.S.

Shaker 0.1499 0.1712 N.S.

Shal 0.0214 0.0243 N.S.

eag 0.4679 0.4347 N.S.

slo 0.4965 0.4846 N.S.

SK 0.7547 0.7700 N.S.

Shaker 0.1114 0.1273 N.S.

Shal 0.0228 0.0276 N.S.

eag 0.4112 0.4287 N.S.

slo 0.3831 0.5448 N.S.

SK 0.4500 0.6058 *

Shaker 0.1832 0.2020 N.S.

Shal 0.0402 0.0453 N.S.

eag 0.2311 0.2906 N.S.

slo 0.5216 0.4336 N.S.

SK 0.7626 0.7713 N.S.

Shaker 0.1202 0.1407 Trend (p = 0.06)

Shal 0.0274 0.0265 N.S.

eag 0.3955 0.4017 N.S.

mRNA levels compared in control v. RNAi knockdown

N.S. indicates No Significance; Trend indicates 0.10>p>0.05; and *

indicates p<0.05.

Control v. Shal

DICER RNAi

Control v. eag

RNAi

Control v. slo

RNAi

Control v. slo 1

mutant RNAi

Control v. SK

RNAi

Control v.

Shaker RNAi