Table 2. Ca²⁺ regulatory effects on Nav1.4 and Nav1.5 studied in heterologous expression systems.

Experimental platform	Pipette buffer (mM concentrations unless otherwise stated)1		Shifts2 due to applied Ca2+				Shifts2 due to calmodulin (CaM)
	0 [Ca2+]	X [Ca2+]	Activation		Inactivation		Activation		Inactivation
			I Na.max	V 1/2	V 1/2	τfast	I Na.max	V 1/2	V 1/2	τfast
Nav1.5  (tsA201; Tan et al. [15])	10 EGTA	1 µM Ca2+ (1.0 EGTA/0.9 CaCl2)3	NIL	-	NIL	?Reduced	NIL	-	?Depol	Reduced
Nav1.4  (HEK293; Deschenes et al.  [33])	10 BAPTA4		-	-	-	-	-	NIL5	NIL5	NIL5
	0 BAPTA	504 nM Ca2+ (3.7 CaCl2/5 BAPTA)4	-	NIL	?Depol	-	-	NIL	Hyper6	NIL
Nav1.5  (HEK293; Deschenes et al.  [33])	0 BAPTA	504 nM Ca2+ (3.7 CaCl2/5 BAPTA)4	-	-	-	NIL	-	NIL	NIL	NIL
Nav1.5  (tsA201; Wingo et al. [7])	20 BAPTA	0–250 nM Ca2+ (0–13.4 CaCl2/20 BAPTA). 1 µM and 10 µM Ca2+ (0.9 CaCl2 or 1.0 CaCl2/1.0 BAPTA)7		NIL	Depol8	NIL	-	-	NIL	-
Nav1.4  (CHO-K1; Young and  Caldwell [34])	5 EGTA		-	-	-	-	NIL	Hyper	Hyper9/NIL10	NIL
Nav1.4  (CHO-K1; Young and  Caldwell [34])		10 µM Ca2+ (5 EGTA/4.9 CaCl2)11, 12	NIL	NIL	NIL	NIL	-	Hyper	NIL	NIL
Nav1.4  (HEK293; Young and  Caldwell [34])	5 EGTA	10 µM Ca2+ (5 EGTA/ 4.9 CaCl2)	-	-	-	-	-	NIL	NIL	NIL
Nav1.5  (CHO-K1; Young and  Caldwell [34])	5 EGTA	10 µM Ca2+ (5 EGTA/4.9 CaCl2)13	NIL	NIL	NIL	NIL	-	Hyper	NIL	NIL
Nav1.4  (tsA201; Shah et al. [13])	20 BAPTA	1 µM Ca2+ (1.0 BAPTA/0.9 CaCl2)	-	-	Depol14	-	-	-	-	-
Nav1.5  (HEK293; Biswas et al.  [35])	20 BAPTA	10 µM Ca2+ (1.0 BAPTA/1.0 CaCl2) 16	NIL	NIL	Depol	Increased	NIL15	NIL15	Depol15	-
Nav1.5  (HEK293; Biswas et al.  [35])		0.5 µM Ca2+ (5 BAPTA/ 4 CaCl2)16					NIL15	NIL15	NIL15	-
Nav1.5  (tsA201; Potet et al. [99])	20 BAPTA	10 µM Ca2+ (1.0 BAPTA/1.0 CaCl2)	-	-	Depol17	NIL
Nav1.5  (tsA201; Chagot et al. [10])	20 BAPTA	1 µM Ca2+ (1.0 BAPTA/0.9 CaCl2).			Depol18
Nav1.5  (tsA201; Sarhan et al. [17])	10 BAPTA	10 µM Ca2+ (1.0 BAPTA/1.0 CaCl2)	-	-	Depol19	NIL	-	-	-	-
Nav1.4  (HEK293; Ben-Johny et al.  [36])	10 BAPTA	10 µM Ca2+ (10 HEDTA/5 CaCl2)	Reduced	-	NIL	-	-	-	-	-
Nav1.4  (HEK293; Ben-Johny et al.  [36])	0.5 EGTA	Activation of co-expressed Cav2.1	Reduced	-	-	-	-	-	-	-
Nav1.4  (HEK293; Ben-Johny et al.  [36])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)21	Reduced	-	NIL	-	Reduced20	-	-	-
Nav1.5  (HEK293; Ben-Johny et al.  [36])	10 BAPTA	10 µM Ca2+ (10 HEDTA/5 CaCl2)	NIL	-	NIL	-	-	-	-	-
Nav1.5  (HEK293; Ben-Johny et al.  [36])	0.5 EGTA	Activation of co-expressed Cav2.1	NIL	-	NIL
Nav1.5  (HEK293; Ben-Johny et al.  [36])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)	NIL	-	NIL
Nav1.4  (glt skeletal muscle cells;  Ben-Johny et al. [36])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)	Reduced	-	-	-	-	-	-	-
Nav1.5  (guinea-pig ventricular  myocytes; Ben-Johny et al.  [36])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)	NIL	-	-	-	-	-	-	-
Nav1.5 with Nav1.4  C-terminal  (HEK293; Yoder et al. [38])	0.5 EGTA	Activation of co-expressed Cav2.1	Reduced22	-	-	-	Reduced	-	-	-
Nav1.5 with Nav1.4  C-terminal  (HEK293; Yoder et al. [38])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)23	Reduced	-	NIL		Reduced
Nav1.4 with Nav1.5  C-terminal  (HEK293; Yoder et al. [38])	0.5 EGTA	Activation of co-expressed Cav2.1	NIL24	-	-	-	-	-	-	-
Nav1.4 with Nav1.5  C-terminal  (HEK293; Yoder et al. [38])	Ca2+ uncaging; 1.0 citrate	0.5–2 µM Ca2+ (1.0 DMN/0.7 CaCl2) 2–8 µM Ca2+ (2 DMN/1.4 CaCl2)	NIL24	-	NIL
Nav1.5  (rabbit ventricular  myocytes; Casini et al. [40])	10 BAPTA	100 nM Ca2+ (CsCl/10 BAPTA)	NIL	NIL	NIL	NIL	-	-	-	-
Nav1.5  (rabbit ventricular  myocytes; Casini et al. [40])		500 nM Ca2+ (CsCl/10 BAPTA)	Reduced	NIL	NIL	NIL	-	-	-	-
Nav1.5  (tsA201; Johnson et al. [18])	20 BAPTA	1.6 µM Ca2+ (5 HEDTA/0.9 Ca2+)							NIL	Increased25

¹∼100 mM F^--containing pipette solutions except: Deschenes et al. [33] apart from C2C12 experiments (Sarhan et al. [17]; Ben-Johny et al. [36]; Yoder et al. [38]; Casini et al. [40]). DMN = DM Nitrophen.

²Key: - = not studied; Nil = no effect; depol = depolarizing; hyper = hyperpolarizing shifts in V_1/2;

³Experiments performed with ±peptide 209–309 (antagonizing Ca²⁺-calmodulin-Nav1.5 binding), I1908E and L1912R IQ mutant and BrS mutant A1924T (Tan et al. [15]);

⁴Pipette solution Cl⁻ or F⁻ and 0 Ca²⁺ (0 mM BAPTA) or 504 nM Ca²⁺ (3.7 mM Ca²⁺/5 mM BAPTA) gave similar results; further 10 µM KN92/KN93 and 100 nM CaMKII inhibitory autocamtide-2 (AIP) controls included;

⁵Effects of 0 Ca²⁺ and of calmodulin-1234;

⁶Double alanine IQ mutation hyperpolarized inactivation V_1/2 and reduced decay constant relative to WT regardless of calmodulin mutation (Deschenes et al. [33]);

⁷Experiments performed ± peptide 209–309 (antagonizing Ca²⁺-calmodulin-Nav1.5 binding;)

⁸Depolarizing effect observed at >150 nM, saturated at 1 µM Ca²⁺, attenuated by EF hand D1790G LQT3 mutation, and abolished by 4× EF hand mutation (Wingo et al. [7]);

⁹Effects of 0 Ca²⁺;

¹⁰Effects of calmodulin-1234;

¹¹Experiments with 10 µM KN93/KN92, N- and C- terminal calmodulin mutants and Nav1.4/Nav1.5 C-terminal chimeras included;

¹²IQ mutations I1727E and L1736R, showed unchanged channel properties relative to WT; I1727E blocked all effects of calmodulin and calmodulin-1234;

¹³Experiments with 10 µM KN93/KN92, N- and C- terminal calmodulin mutants and Nav1.4/Nav1.5 C-terminal chimeras included (Young and Caldwell [34]);

¹⁴Single, A1924T, but not double IQ mutation also caused depolarizing V_1/2 shift (Shah et al. [13]);

¹⁵Studies with calmodulin1234 included;

¹⁶Ca²⁺ hyperpolarized inactivation V_1/2 both in mutants lacking C-terminal and double alanine IQ mutation. Both EF hand LQT3 mutation D1790G and 4X mutation hyperpolarized inactivation V_1/2 but were unresponsive to Ca²⁺ (Biswas et al. [35]);

¹⁷A1924T mutant showed difference from WT only at 0 Ca²⁺ (Potet et al. [99]);

¹⁸EF-2X mutation caused hyper and abolished Ca²⁺ action (Chagot et al. [10]);

¹⁹No effect at 0.3 µM Ca²⁺ (Sarhan et al. [17]);

²⁰Time constants of Ca²⁺ dependent inactivation onset reported for different [Ca²⁺];

²¹Double alanine IQ mutation caused Ca²⁺ dependent facilitation; myotonia mutants Q1626E and F1698I showed attenuated Ca²⁺-dependent inhibition and lesser reduction in I_max than WT. EF hand, D1621A and D1623A, mutations had no effect (Ben-Johny et al. [36]);

²²WT calmodulin and calmodulin-34 maintained Ca²⁺ dependent inactivation, calmodulin-12 resulted in loss of such inactivation.;

²³Nav1.5 mutant without the post IQ motif showed persistent Ca²⁺ dependent inhibition;

²⁴Ca²⁺ dependent inactivation persisted with Nav1.5 C-terminal domain lacking post IQ segment (Yoder et al. [38]);

²⁵Ca²⁺-calmodulin (but not apo-calmodulin) binding implicated in slowed kinetics of inactivation and accelerated recovery from inactivation, but not in Nav1.5 double mutants involving both sites A and B of II–III linker region.