Evidence for a fast non-selective low voltage-activated cationic channel in rat pulmonary vein cardiomyocytes

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Evidence for a fast non-selective low voltage-activated cationic channel in rat pulmonary vein cardiomyocytes

C. O. Malécot

To cite this version:

C. O. Malécot. Evidence for a fast non-selective low voltage-activated cationic channel in rat pul- monary vein cardiomyocytes. 30th Ion Channel Meeting Association Canaux Ioniques, Sep 2019, Sète, France. www.canaux-ioniques.fr, 2019, 30th Ion Channel Meeting Association ”Canaux Ioniques” - Program & Abstracts. �hal-02458570�

Evidence for a Fast Non-selective

Low Voltage-activated Cationic Channel in Rat Pulmonary Vein Cardiomyocytes

Claire O. Malécot

Laboratoire Signalisation et Transports Ioniques Membranaires, EA 7349,

Universités de Tours et de Poitiers, Faculté des Sciences, Parc de Grandmont, 37200 TOURS

Ectopic foci in pulmonary veins (PV) myocardial sleeves are involved in the onset of atrial fibrillation. Although T-type Ca channels exist in rat PV cardiomyocytes (CM), we found an additional fast low voltage-activated (FLVA) Ca current, susceptible to trigger ectopic foci. The aim of this study was to characterize the underlying channel selectivity and pharmacology.

Introduction

PVCM were enzymatically isolated from the rat main PVs with liberase and protease. Whole- cell I

_Ca

(2 or 5 mM extracellular Ca) was recorded by applying voltage steps from -100mV and with classical Na- and K-free bath solutions (TEA-Cl based, pH 7.4). Pipette solution was Cs-aspartate based and contained Mg-ATP, Tris-phosphocreatine and EGTA (pH 7.25).

Methods

V_o = (RT/F) log_e {([X]_o + 4 (P'_Ca/P_X) [Ca]_o) / ([X]_i + 4 (P'_Ca/P_X) [Ca]_i exp(FV_o/RT))}

- Davies equation for activity coefficient _i : log₁₀ _i = -0.509 z_i² (( I^1/2 /(1 + I^1/2)) – 0.3 I ) where I is the ionic strength and z the ion valence

- With P'_Ca = P_Ca / ((1+exp(FV/RT)), the reversal potential V_o of the current in bi-ionic conditions (Ca²⁺, X⁺) is given by the following modified GHK equation:

Figure 5. Aa and Ba, effects of 5µM Nifedipine (Nif) and 0.3 µM BayK8644 (BayK). Ab and Bb, Mean IV curves in control conditions (5 mM Ca²⁺

+ 5mM K⁺ to have a larger control current) and in the additional presence of 5 µM Nif (n=5) or 0.3 µM BayK (n=-6), respectively. (*): P< 0.05 vs.

control.

Figure 3. A, External Ca²⁺-dependence of the FLVA current. B and C, effects of Sr²⁺ (B) or Ba²⁺ (C) equimolar substitution for Ca²⁺ on the current. In Cb, Ba²⁺ substitution for Ca²⁺ reveals a slower activating and inactivating current component.

Ca

B

0

40 pA 10 ms 0 Ca²⁺

5 mM Ca²⁺

5 mM Sr²⁺

100 pA 20 ms

0

0

5 mM Ca²⁺

A

b

Figure 3

5 mM Ba²⁺

5 mM Ca²⁺

0

20 ms 100 pA

5 mM Ba²⁺

5 mM Ca²⁺

20 ms 50 pA

-40 mV -35mV

-35 mV -35 mV

Divalent cations

Figure 4. Na⁺ (Aa) and K⁺ (Ba) permeation in the presence of 5 mM Ca². Ab and Bb, mean IV curves. Na⁺: (, ), n=6;

(), n=5. K⁺: n=8 for each curve. C, channel conductance to Na⁺ or K⁺ in the presence of 5 mM Ca²⁺. Smooth line represents adjustment of the Langmuir isotherm equation to the mean data points (same n values as above).

0 5 mM Ca²⁺

200 pA 10 ms 0

5 mM Ca²⁺

+3 mM Na⁺ +1 mM Na⁺

-80 -60 -40 -20 0

-16 -12 -8 -4 0

Control +1 mM Na⁺

+3 mM Na⁺ _pA

/pF

mV

200 pA 10 ms +5 mM K⁺

+10 mM K⁺

-80 -60 -40 -20 0

-12 -8 -4 0

Control +5 mM K⁺

+10 mM K⁺ pA/pF mV

0 2 4 6 8 10 12

0.0 0.1 0.2 0.3 0.4 0.5

Ca²⁺ + Na⁺ Ca²⁺ + K⁺

b

C Ba

Ion channel conductance (nS/pF)

Monovalent cation added (mM) 5 mM Ca²⁺

Aa b

Figure 4

-40 mV

-35 mV

Monovalent cations

-35 mV

-30 mV -35 mV

Dihydropyridines sensitivity

b Ba

50 pA 20 ms

Control 5 µM Nif 0

Aa b

-80 -60 -40 -20 0

-6 -4 -2 0

Control +5 µM Nif

* *

* *

* *

* *

pA/pF

mV

*

Figure 4

Control 20 ms 25 pA 0

0.3 µM BayK

-80 -60 -40 -20 0

-24 -21 -18 -4 -2 0

Control

+0.3 µM BayK

*

* *

*

pA/pF

mV

-35 mV

-40 mV

Pharmacology of FLVA current

Control current was recorded in the presence of either 2 or 5 mM CaCl₂. 5 mM KCl was added to the solution for Nifedipine and BayK experiments to increase the current amplitude. P values for paired t-test except last compound (Mann-Whitney Rank Sum test)

Conclusions

The results suggest that the FLVA channel is neither a Ca ²⁺ nor a Na ⁺ channel but more likely a fast voltage-gated non-selective cationic channel with a dihydropyridine binding site and unique properties.

FLVA channel, a Ca ²⁺ channel???

For Against

Ca

²⁺

permeation Ba

²⁺

, Sr

²⁺

non permeant

Sensitivity to DHP, Co

²⁺

No block by 0.2 µM diltiazem Sensitivity to β-stim Reversal potential

Na

⁺

, K

⁺

and Cs

⁺

permeation P'

_Ca

> P

_Na

or P

_K

FLVA channel, a Na ⁺ channel???

For Against

Na

⁺

permeation Reversal potential

Sensitivity to TTX Ranolazine-insensitive Ba

²⁺

, Sr

²⁺

non permeant K

⁺

and Cs

⁺

permeation

Sensitivity to DHP P'

_Ca

> P

_Na

Ion Permeation

Pharmacology

V_rev: current apparent reversal potential. P'_Ca/P_X: relative permeabilty of Ca²⁺ over cation X⁺. Ionic strength I of the extracellular recording solution was 0.162 mol/L in 5 mM CaCl₂ control conditions. With 1, 3, 5 and 10 mM added XCl (with X representing Na⁺ or K⁺), I increased to 0.163, 0.165, 0.167 and 0.172 mol/L, respectively.

FLVA channel selectivity

50 pA 10 ms

  

20 ms 25 pA

0 0

0

20 ms 25 pA



-80 -60 -40 -20

-5 -4 -3 -2 -1 0

c

pA/pF

mV

Figure 1

Aa b

B

-35 mV -40 mV -35 mV

Figure 1. Aa, fast activating and inactivating (FLVA) current; Ab, slow activating and inactivating current (SLVA); Ac, fast activating and slow inactivating current (FSLVA). The symbols above each trace refer to the mean current voltage (IV) relationships shown in B. Data points are mean±SE of n PVCM. (), n=12; (), n=37; (), n=10.

Low voltage activated (LVA) Ca

²⁺

currents from -100 mV in PVCM in the presence of 5mM Ca

²⁺

0 +10 µM TTX

25 pA 10 ms

0 0

5 mM Ca²⁺

+10 µM TTX

5 mM Ca²⁺

20 ms 25 pA

+10 µM Rano 5 mM Ca²⁺

25 pA 20 ms

-80 -60 -40 -20

-4 -3 -2 -1 0

5 mM Ca²⁺

+10 µM TTX

b Ba

pA/pF

mV

Aa _b

c

Figure 3

-80 -60 -40 -20

-3 -2 -1 0

5 mM Ca²⁺

+10 µM Rano

pA/pF

mV

Figure 2. A, effects of 10 µM TTX on the FLVA current. a, complete block and b, unveiling of a slower activating and inactivating Ca²⁺ current component. c, mean IV curves, n=8. B, lack of effect of 10 µM ranolazine (Rano) in the presence of 5 mM Ca²⁺. b, Mean IV curves in control conditions and in the presence of 10 µM Rano (n=6 for each curve)

TTX-sensitive, ranolazine-insensitive FLVA current

FLVA current: general

V_rev: current apparent reversal potential. P values for Mann-Whitney Rank Sum test. (*) V_rev was too small in 5 PVCM to be accurately determined

P' _Ca / P _Na = P' _Ca / P _K  2

FLVA-I _Ca

 found in only ~53.5 % of PVCM

 apparent V_rev follows Nernst eq. between 2 and 5 mM Ca²⁺

 looks like I_Ca,TTX

 BUT…..