A R T I C L E S

Life-threatening arrhythmias arise from acquired1 and inherited2,3

Na+ channel dysfunction. Several mutations have been identifiedwithin the C-terminal region of hH1 that provoke arrhythmia syn-dromes by altering the channel gating function4–7. One of these muta-tions, D1790G, has been associated with the Na+ channel–linked formof the congenital long QT syndrome (denoted LQT3)8,9. Althoughrecent analysis of the hH1 C terminus revealed an ordered, helicaldomain10, assigning structure and function to localized regions hasproven difficult. Here we report the identification of a structural motifthat underlies the pathophysiology of LQT3 through complementaryapplication of sequence analysis, patch-clamp electrophysiology andbiophysical analysis of a bacterially expressed C-terminal fragment.These results also provide an improved understanding of the func-tional role of the Na+ channel C terminus in cardiac rhythm.

RESULTSIdentification of an EF-hand in hH1Analysis of the hH1 C terminus using PSI-BLAST11,12 (Fig. 1a) revealedhomology to EF-hand helix-loop-helix motifs in a number of Ca2+-binding proteins, including calmodulin (CaM). EF-hand proteins medi-ate the transduction of Ca2+ signals in cellular responses, includingregulation of ion channels13. Notably, the EF-hand was found ∼ 120 residues upstream from an ‘IQ-domain’ calmodulin-binding siteidentified previously (residues 1909–1919)14,15. A structure-based mul-tiple sequence alignment with several of the highest scoring hits from thePSI-BLAST search, all proteins with EF-hands whose three-dimensionalstructures are known (PDB entries 1C07, 1C7V, 1EXR, 1BJF and 1TRF;Fig. 1a), provided further support for this structural class assignment.

A single EF-hand motif is not an intrinsically stable structure, and isalways found in larger aggregates16,17. A pair of EF-hands forms theminimal stable structural unit—a four-helix bundle domain18,19. The hH1 C terminus has a pair of EF-hands in the regionGlu1773–Asp1852. There is a Ca2+-binding site in the first EF-handsequence, based on the close fit to the consensus binding loop(Fig. 1a). The second EF-hand loop is not expected to bind Ca2+

because it lacks key consensus side chains involved in chelating the ion.The lack of a competent Ca2+ site in the second EF-hand (Fig. 1a) isnot atypical, and binding of a single Ca2+ ion is sufficient to activateseveral EF-hand Ca2+ sensor domains (such as the N-terminal domainof cardiac troponin C and the C-terminal domain of centrin).

Threading calculations for hH1(1773–1852) against a library of allstructurally characterized tertiary fold templates confirmed that apaired EF-hand motif is a highly plausible three-dimensional structurefor this sequence. A structural model (Fig. 1b,c) was therefore con-structed using a structure-based sequence alignment with the five EF-hand protein templates listed in Figure 1a. The presence of anordered structure is consistent with the fact that the C terminus contains helical secondary structure10.

Ca2+ modulates hH1 inactivationLike most voltage-gated ion channels, hH1 can occupy an open (Na+-conducting) state, as well as two distinct nonconducting states,termed ‘closed’ and ‘inactivated.’ Channels in the closed state can openand generate Na+ current (INa) in response to a stimulus, whereaschannels in the inactivated state cannot open and are functionallyunavailable. Under resting conditions, the potential across the

Departments of 1Pharmacology, 2Biochemistry, 3Medicine, 4Chemistry, 5Physics and 6Anesthesiology and 7Center for Structural Biology, Vanderbilt University,Nashville, Tennessee 37232, USA. 8These authors contributed equally to this work. Correspondence should be addressed to W.J.C. (walter.chazin@vanderbilt.edu) or J.R.B. (jeff.balser@vanderbilt.edu).

Published online 22 February 2004; doi:10.1038/nsmb737

An EF-hand in the sodium channel couples intracellularcalcium to cardiac excitabilityTammy L Wingo1,8, Vikas N Shah2,7,8, Mark E Anderson1,3, Terry P Lybrand1,4,7, Walter J Chazin2,5,7 & Jeffrey R Balser1,6

Sodium channels initiate the electrical cascade responsible for cardiac rhythm, and certain life-threatening arrhythmias arisefrom Na+ channel dysfunction. We propose a novel mechanism for modulation of Na+ channel function whereby calcium ionsbind directly to the human cardiac Na+ channel (hH1) via an EF-hand motif in the C-terminal domain. A functional role for Ca2+

binding was identified electrophysiologically, by measuring Ca2+-induced modulation of hH1. A small hH1 fragment containingthe EF-hand motif was shown to form a structured domain and to bind Ca2+ with affinity characteristic of calcium sensorproteins. Mutations in this domain reduce Ca2+ affinity in vitro and the inactivation gating effects of Ca2+ in electrophysiologyexperiments. These studies reveal the molecular basis for certain forms of long QT syndrome and other arrhythmia-producingsyndromes, and suggest a potential pharmacological target for antiarrhythmic drug design.

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 3 MARCH 2004 219

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

membrane determines the ratio of closed to inactivated channels in apopulation of Na+ channels. Depolarization of the membrane poten-tial shifts the balance to favor the inactivated state. Because this ‘avail-ability curve’ (Fig. 2a) is steepest at voltages that approach the cardiacmyocyte maximum diastolic membrane potential (Vrest of about–90 mV), the population of Na+ channels available to open is sensitiveto even modest changes in Vrest. Pathologic conditions, such asischemia or acidosis, which alter Vrest, can markedly change hH1 avail-ability, modifying INa and destabilizing the cardiac rhythm20.

The potential for a functional EF-hand domain in hH1 led us toinvestigate the effects of intracellular Ca2+ on the voltage dependenceof Na+ channel availability (Fig. 2). For these studies, whole-cell Na+

currents (INa, Fig. 2a) were recorded in voltage-clamped tsA201 cells transiently transfected with wild-type hH1 cDNA. Voltage-

dependent channel availability was assessedby quantifying INa during an activating pulseto –20 mV (Fig. 2a) as a function of various‘test’ resting membrane voltages. Figure 2aindicates that raising free Ca2+ concentra-tions from 0 to 10 µM caused a rightwardshift in availability, with the effect saturatingat ∼ 1 µM. The membrane potential at which50% of the Na+ channels were available toopen (V1/2) was: –98.3 ± 1.54 (n = 11), –95.4± 0.79 (n = 4), –92.8 ± 1.07 (n = 8, P < 0.05),–91.6 ± 0.9 (n = 8, P < 0.01), –87.8 ± 1.6 (n =7, P < 0.001) and –87.1 ± 1.03 mV (n = 17, P < 0.0001) for 0, 100, 150 and 250 nM and 1and 10 µM Ca2+, respectively. Slope factor (k)in zero free Ca2+ decreased slightly in 10 µMCa2+: 6.38 ± 0.24 (n = 11) versus 5.54 ± 0.23(n = 17), respectively (P < 0.05 (N.S., not sig-nificant)). Significant differences indicatecomparisons to the 0 Ca2+ condition. Theobserved change is consistent with a destabi-lizing effect on inactivation gating. A dose-response curve fitted to the V1/2 data (Fig. 2c)yields EC50 values, with and without theCaM inhibitory peptide 290–309, of 175 and199 nM, respectively. These values are consis-tent with a physiological and structural rolefor Ca2+ binding to the EF-hand18. Othergating processes, including activation andrecovery from inactivation, were evaluatedfor wild-type channels as described21, anddid not respond to added Ca2+. Activationcurve Boltzmann parameters for wild-typedata in zero free Ca2+ (20 mM BAPTA) and10 µM Ca2+ were: V1/2 = –42.63 ± 2.3 (n = 7),k = 7.53 ± 0.578 (n = 7), and V1/2 = –42.61 ±3.1 (n = 8), k = 6.43 ± 0.702 (n = 8), respec-tively; P = N.S. for both V1/2 and k values.The kinetics of recovery from inactivationwere also compared, giving values of: A1 =0.696 ± 0.014, A2 = 0.241 ± 0.017, τ1 = 6.84 ±0.638, τ2 = 125.3 ± 31.1, n = 6 for zero freeCa2+; and A1 = 0.764 ± 0.038, A2 = 0.250 ±0.025, τ1 = 5.73 ± 0.807, τ2 = 135.5 ± 20.6, n = 4 for 10 µM Ca2+ (see Methods). P = N.S.for all parameter comparisons (low versushigh Ca2+).

Although effects of Ca2+-dependent cofactors on Na+ channel gatinghave been identified14,15, direct effects of Ca2+ on Na+ channel avail-ability have not been described. An important change in the presentstudies was the use of BAPTA as a Ca2+ buffer in the patch electrodesolution. Over the range of free Ca2+ concentrations tested (0 to10 µM), the V1/2 values for the hH1 availability curve (–87 to –98 mV)bracket the value obtained in earlier studies using 10 mM EGTA and noadded Ca2+ (–93.5 mV)14,15. In fact, the V1/2 of the availability curve innominally Ca2+-free solutions containing EGTA is quite close to theV1/2 measured at the EC50 Ca2+ concentration when BAPTA is used,suggesting EGTA is not as effective as BAPTA in maintaining Ca2+ atlow (nanomolar) levels. Hence, with EGTA as the buffer, detection ofCa2+ effects on Na+ channel availability would be difficult, since theV1/2 dynamic range would be no greater than 5 mV (from –93 to

220 VOLUME 11 NUMBER 3 MARCH 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

Figure 1 Structure-based sequence alignment and homology modeling of the proximal region of thehH1 C terminus. (a) hH1-cterm (hH1 residues 1773–1852) was aligned with Ca2+-binding proteinsidentified using PSI-BLAST (PDB entries 1C07 (human Eps15), 1C7V (Ca2+-bound C-terminal domainof calcium vector protein), 1EXR (Ca2+-bound calmodulin), 1BJF (neurocalcin), and 1TRF (troponinC)). Regions highlighted in black indicate key residue locations normally responsible for Ca2+ ioncoordination (designated as positions 1, 3, 5 and 12) in a pair of putative EF-hand loops. Numberslocated above each main box identify the location of each amino acid in the alignment, but do not referto the location of the amino acid in the protein. The region highlighted in blue identifies an EF-handpredicted to have a strong Ca2+-binding site, whereas the region highlighted in red identifies a secondEF-hand that could serve to stabilize the requisite paired structure. Asterisk indicates ten amino acidsomitted after alignment owing to space constraints (1BJF residues omitted are VSSVMKMPED). (b) Energy-minimized three-dimensional model of the Na+ channel C terminus (green ribbon)superimposed on the backbone structure of the Ca2+-binding proteins (red, used as templates in modelconstruction). (c) Predicted model for the hH1 proximal C terminus highlighting the features of the EF-hand loop region. The proximal, functional EF-hand loop is yellow, and the distal EF-hand loop isorange. Key residues are color-coded for clarity and shown in ball-and-stick representation. The redresidue represents position 3 for Ca2+ ion coordination, and is also a residue mutated in long QTsyndrome (D1790G). Other residues important for Ca2+ ion coordination (position 1, Glu1788; position5, Asp1792; position 12, Glu1799) are highlighted in white. Additional positions where mutationselicit arrhythmia phenotypes (Glu1784, Phe1795, see text) are green. A translucent cyan sphererepresents the Ca2+ ion location in the EF-hand.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

–98 mV). Overall, our findings suggest that the selection of metal ionbuffer is critical when evaluating the function of the hH1 EF-hand incellular preparations.

In earlier work describing Ca2+-CaM mediated effects on Na+ channelinactivation, we and others have noted changes in the rate of fast Na+ cur-rent decay that appear to be mediated by Ca2+ (refs. 14,15). However,with BAPTA substituted for EGTA, we find the hH1 INa decay rate(Fig. 2a) does not change appreciably with added Ca2+; τ was 0.84 ±0.06 ms (n = 5) and 0.71 ± 0.01 ms (n = 5) in 0 and 10 µM free Ca2+,respectively (P = N.S). Recognizing that BAPTA provides more stringentCa2+ buffering conditions, and more rapid Ca2+-binding kinetics22, wespeculate that the small Ca2+-dependent effect previously noted on hH1INa decay14 may be caused by activation of Ca2+-signaling pathways thatmodulate hH1, but that are less tightly controlled by EGTA than BAPTA.

Ca2+ modulation is independent of CaM and �1Earlier studies examining ancillary β subunit (β1) modulation of hH1report diverse effects. Here, coexpression of β1 caused an overall depo-larizing shift in the availability curve, as reported by other investigatorsusing analogous experimental conditions8,9. Nonetheless, with β1coexpressed, raising intracellular Ca2+ caused an additional depolariz-ing shift: V1/2 was –91.6 ± 1.67 mV in Ca2+-free conditions (n = 4),versus 78.9 ± 1.9 mV in 10 µM Ca2+ (n = 5), P < 0.002 (data notshown). Hence, although β1 modifies hH1 availability, it does notinfluence the Ca2+-dependent component of hH1 availability.

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 3 MARCH 2004 221

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

cur

rent

Membrane potential (mV)

290–309

100101 1,000 10,000–102

–100

–98

–96

–94

–92

–90

–88

–86

–84

V1/

2

[Ca2+] (nM)

290–309Peptide-free

0.0

–160 –140 –120 –100 –80 –60 –40 –160 –140 –120 –100 –80 –60 –40

0.2

0.4

0.6

0.8

1.0

2 ms

2 nA

0 mV Ca2+-free

2 nA

10 µM Ca2+

0 Ca2+

100 nM Ca2+

150 nM Ca2+

250 nM Ca2+

1 µM Ca2+

10 µM Ca2+

500 ms

Peptid e-free

–160 mV

–120 mV

–40 mV

–20mV20 ms

Nor

mal

ized

cur

rent

Membrane potential (mV)

b

c

a

Figure 2 Voltage-dependent availability of hH1 is Ca2+ sensitive. (a) Theratio of the INa magnitude (at –20 mV) associated with each test potential,relative to that associated with the most negative test potential (–160 mV,where no channels inactivate). The period at each test voltage was 500 ms,allowing rapid inactivation gating processes to reach steady state, whileavoiding ultraslow components of inactivation14. Solid lines indicate a least-squares fit of a Boltzmann function to the data. Inset, representative INameasured at –20 mV after holding at –120 mV in 0 and 10 µM free Ca2+

(see Methods). INa decay was fitted using a single exponential function (see Methods). Intracellular Ca2+ did not alter the rate of current decay. (b) Voltage dependence of inactivation dose-response analysis with CaMinhibitory peptide 290–309, analyzed as in panel a. (c) Dose-responseanalysis of data represented in Figure 2a,b.

To exclude the possibility that the Ca2+-dependent changes in hH1availability are related to the downstream IQ domain14,15, we used aselective peptide CaM antagonist (290–309) that inhibits Ca2+-dependent CaM binding to hH1 (refs. 14,15). We find no effect of theinhibitory peptide on the voltage dependence of channel availability,or on the sensitivity of this gating process to Ca2+ (Fig. 2b,c). The datayielded the following V1/2 values: –97.6 ± 1.9 (n = 4), –93.7 ± 1.8 (n = 4), –90.5 ± 0.69 (n = 4, P < 0.05), –91.6 ± 0.94 (n = 4, P < 0.05),–87.3 ± 0.98 (n = 3, P < 0.01), –85.3 ± 0.76 (n = 6, P < 0.001) for 0, 100,150 and 250 nM and 1 and 10 µM intracellular free Ca2+, respectively.Significant differences indicate comparisons to the 0 Ca2+ condition.These data suggest that hH1 Ca2+-dependent availability (Fig. 2a) isnot mediated by CaM14,15.

hH1-EF has a functional Ca2+ binding siteTo assess whether the consensus EF-hand motif is capable of bindingCa2+, we characterized a bacterially expressed construct (hH1-EF) con-taining the first 148 residues of the C-terminal region. Circular dichro-ism (CD) was used to examine secondary structure, NMR to probetertiary structure, and fluorescence spectroscopy to directly monitor thebinding of Ca2+. The profile of the CD spectrum of hH1-EF has charac-teristic double minima at 208 and 222 nm, indicative of substantial heli-cal content (data not shown). Like many EF-hand proteins, formation ofa well-defined tertiary structure is dependent on Ca2+; the NMR signalsof hH1-EF in the absence of Ca2+ are much less dispersed and consider-ably broader, indicating that the tertiary structure is not fully stabilized(that is, the conformation of the protein is heterogeneous). Similarobservations have been made for a variety of EF-hand proteins such asthe C-terminal domain of sarcoplasmic calcium-binding protein23,

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

centrin24 and calcium vector protein25. Upon addition of Ca2+, NMR signals narrow and are much more widely dispersed across theNMR spectrum, particularly in the 1H dimension. The dispersion of 1Hsignals from 6.5-11.5 p.p.m. in the 2D 15N-1H HSQC NMR spectrum ofCa2+-loaded hH1-EF (Supplementary Fig. 1 online) is clear evidence of a well-folded structural domain.

Trp1798 in position 11 of the first EF-hand loop (Fig. 1a) serves as aconvenient fluorescent probe to monitor Ca2+ binding as this is the onlytryptophan in hH1-EF. A substantial (∼ 20%) quenching of fluorescenceaccompanied by a small blue shift in the spectrum is observed upon

addition of Ca2+ to the protein (Fig. 3b). Fitting a plot of fluorescenceintensity versus Ca2+ concentration provides an estimate of the Ca2+

dissociation constant (Kd) of 1.3 µΜ, within in the range expected for aCa2+ sensor domain in vitro18.

EF-hand mutations alter the Ca2+ effectThe LQT3 mutation D1790G is located at a position where the nativeaspartic acid side chain directly coordinates Ca2+ in consensus EF-hand loops (position 3; Fig. 1a,c). Based on extensive earlier muta-tional analysis on EF-hand proteins18, the D1790G mutation would be

222 VOLUME 11 NUMBER 3 MARCH 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

0.6

0.7

0.8

0.9

1

330 340 350 360 370

Wavelength (nm)

Nor

mal

ized

fluo

resc

ence

inte

nsity

WT, 0 Ca2+

WT, 20:1 Ca2+/protein

D1790G, 0 Ca2+

D1790G, 20:1 Ca2+/protein

4x, 0 Ca2+

4x, 20:1 Ca2+/protein

[Ca+2]/[protein] ratio

Nor

mal

ized

fluo

resc

ence

inte

nsity

0.87

0.89

0.91

0.93

0.95

0.97

0 8 16 24 32 40 48 56 64 72

0.82

0.86

0.90

0 100 200 300 400 500 600

1H (p.p.m.)

8.78.99.19.39.59.7

15N

(p.p

.m.)

111

113

115

117

119

121

123

125

111

113

115

117

119

121

123

125

-160 -140 -120 -100 -80 -60 -40

0.0

0.2

0.4

0.6

0.8

1.0

Nor

ma

lized

cur

rent

Membrane potential (m/v)

WT, 0 Ca2+

WT, 10 µM Ca2+

D1790G, 0 Ca2+

D1790G, 10 µM Ca2+

4x, 0 Ca2+

4x, 10 µM Ca2+

0.990.94

0.96

b

d

a

c

Figure 3 Structural characterization and calcium binding to hH1-EF, and electrophysiological characterization of Ca2+-dependent inactivation effects causedby hH1 EF-hand mutations. (a) The downfield-shifted region of wild-type hH1-EF spectrum (dashed contours) acquired at 28 °C superimposed with thecorresponding region of the spectrum, acquired under identical conditions, for the D1790G mutant (solid contours). (b) Calcium dependence of the intrinsictryptophan fluorescence spectrum of hH1-EF (square) and the D1790G (circle) and 4× (triangle) mutants. Filled symbols represent the apo state and opensymbols represent a solution containing ∼ 20:1 Ca2+/protein ratio (by concentration) (c) Plot of fluorescence intensity versus added Ca2+ for hH1-EF (solidline) and the D1790G mutant (dotted line). The line through the points is the best fit to a single-site standard binding curve. Because the binding to themutant is incomplete in the main panel, an inset is included to show the full binding curve (to 600-fold excess). (d) Voltage dependence of availability forD1790G and 4× was analyzed in the manner shown in Figure 2a. For the D1790G mutant, the V1/2 was –101.9 ± 1.7 (n = 4) and –95.8 ± 1.0 mV (n = 7) for 0 Ca2+ and 10 µM Ca2+ respectively (P < 0.01). k values were 7.55 ± 0.14 (n = 4) and 5.68 ± 0.219 (n = 7) for 0 Ca2+ and 10 µM Ca2+, respectively (P < 0.001). The 4× mutant shows V1/2 values of –101.3 ± 2.2 mV in 0 Ca2+ (n = 9) and –101.1 ± 1.1 in 10 µM Ca2+ (n = 9) (P > 0.05; N.S., notsignificant). k values were 7.58 ± 0.43 (n = 9) and 6.28 ± 0.276 (n = 9) for 0 Ca2+ and 10 µM Ca2+, respectively (P < 0.01). Wild-type curves fromFigure 2a (0 free Ca2+ and 10 µM Ca2+) are superimposed on the graph for comparison. The voltage dependence of inactivation for 4× with the CaMinhibitory peptide 290–309 included in the pipette solution (10 µM) was also assessed (data not shown). The V1/2 values were –97.2 ± 1.2 (n = 3) and–100.7 ± 3.1 mV (n = 3) for 0 Ca2+ and 10 µM Ca2+ respectively (P = N.S.). Inset, INa decay was fitted using a single exponential function (see Methods);fitted parameters for τ (ms) were 0.99 ± 0.26 for Ca2+-free conditions (n = 5) and 0.84 ± 0.12 for 10 µM Ca2+ (n = 6) (P = N.S.).

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

predicted to reduce Ca2+ affinity by about one order of magnitude.Such an effect could conceivably alter inactivation gating in hH1, andat least partly explain the LQT phenotype9.

As predicted (Fig. 3d), D1790G significantly impairs the inactivation-destabilizing effect of Ca2+ on hH1. In D1790G, 10 µM Ca2+ shifts theV1/2 positively by only 6.1 mV, versus 11.1 mV in wild type (P < 0.0001; see legends of Figs. 2a and 3d for absolute V1/2 values). Theavailability curves for D1790G and wild type in 0 Ca2+ did not differsubstantially, suggesting that the mutation does not cause an allostericeffect on channel structure. However, in nearly all nonzero Ca2+ con-centrations, the voltage-dependent availability of the mutant is left-shifted (hyperpolarized) relative to wild type (Fig. 3d), as notedpreviously in studies using less stringent Ca2+-buffering conditions, bututilizing β1 subunit coexpression8,9. Here we find that under moretightly controlled Ca2+ buffering conditions, coexpression of β1 is notrequired to identify a hyperpolarizing shift in D1790G availability relative to wild type.

The impact of a hyperpolarizing shift in availability due to D1790Ghas been mathematically modeled in detail8,9. The reduced availabilityof Na+ channels at the resting membrane potential leads to a diminishedNa+ current during the initiation of the action potential, which triggersa cascade of changes in the kinetics of multiple ionic currents during theaction potential. This prolongs the cardiac action potential, increasingthe likelihood of cardiac arrhythmias as seen in LQT3 (refs. 8,9). Arecent study of D1790G has also detected a PKA-sensitive increase insustained, bursting Na+ channel activity during sustained depolariza-tion26, a gating behavior that also provokes LQT32,3. In our studies,addition of 10 µM intracellular Ca2+ did not increase the magnitude ofthe plateau current in either the wild-type (0.24 ± 0.14%, n = 11, plateaucurrent relative to peak inward current) or D1790G (0.24 ± 0.057%, n = 7). Hence, we postulate that D1790G brings about LQT3 by pro-longing the cardiac action potential through two complementary mech-anisms: (i) a protein kinase A-dependent, Ca2+-independent increase inchannel bursting that increases the plateau current26, and (ii) disruptionof Ca2+-binding in the hH1 EF-hand, causing a hyperpolarizing shift inhH1 voltage-dependent availability (Fig. 3d).

Biophysical analysis of hH1-EF containing the D1790G mutationreveals virtually no changes in the NMR and CD spectra.Superimposing the downfield region of the 2D 15N-1H HSQC NMRspectra to compare the mutant and wild-type proteins provides con-vincing evidence that the mutation does not alter the protein structure(Fig. 3a). In contrast, measurements of tryptophan fluorescence showthat Ca2+ binds more weakly to the mutant (Fig. 3b,c) with a Kd of27 µΜ, ∼ 20-fold lower than that of hH1-EF. This reduction in affinityis consistent with the effects of mutations in other EF-hand Ca2+-binding proteins18 and is sufficient to reduce the ability of the EF-handdomain to act as a Ca2+ sensor. Together, the patch-clamp and bio-physical data suggest that D1790G causes LQT3 through reducingCa2+-dependent regulation of hH1 availability by disrupting Ca2+

binding in the EF-hand.

Loss of Ca2+ effect in an EF-hand knockoutTo further test this hypothesis, a mutant hH1 was designed to completelyeradicate Ca2+ binding to the functional EF-hand. The first EF-hand-binding loop contains the consensus acidic side chains at positions 1, 3and 5, and the highly conserved glutamic acid at position 12 (Fig. 1a), allof which directly chelate Ca2+. The corresponding “4×” mutant (E1788AD1790A D1792A E1799A) was prepared and examined electrophysio-logically (whole channel) and in vitro (hH1-EF construct).

Raising free Ca2+ from 0 to 10 µM did not change 4× mutant voltage-dependent availability (Fig. 3d). Moreover, the 4× mutant

availability curve in 0 Ca2+ was similar to that of wild type (Fig. 3d), aswas the INa decay rate (Fig. 3d, inset), suggesting that the quadruplesubstitution does not induce marked structural changes in the C terminus. Like that of wild type, the response of the 4× mutant toCa2+ was not altered by the CaM inhibitory peptide (290–309) (see Fig. 3d legend for data).

The results from biophysical analysis of hH1-EF containing the 4×mutation were fully consistent with the electrophysiological observa-tions. Ca2+ titrations monitored by fluorescence showed almost noresponse, even at 1,000-fold excess of Ca2+ (Fig. 3b), indicating that 4×hH1-EF does not bind Ca2+ with any detectable affinity. The CD spec-trum of 4× hH1-EF confirmed that the helical structure of the wild-type protein was retained, and the NMR spectrum in the absence orpresence of Ca2+ was the same as that of Ca2+ hH1-EF (data notshown). These results, together with the electrophysiologic findings,strongly suggest that the 4× mutation causes loss of Ca2+-bindingactivity without loss of overall structure.

DISCUSSIONCa2+ modulation of ion channelsMultiple sequence alignment, homology modeling and biophysicaldata on hH1-EF constructs leave virtually no doubt as to the presenceof an EF-hand domain in hH1 that binds Ca2+ with affinity well withinthe range of known Ca2+ sensors. The extensive similarity of the NMRspectrum of hH1-EF with that from a construct containing the first95 amino acids (the four helices of the EF-hand domain) of the C-terminal region provides further evidence for the proposed EF-hand domain (V.N.S., K. Weiss and W.J.C., unpublished results).Binding of Ca2+ to this EF-hand domain has a role in a critical hH1gating function: increasing channel activity by inducing a depolarizingshift in the voltage-dependence of channel availability.

Although the effects of mutations in the EF-hand were consistentbetween the electrophysiologic and spectroscopic analyses, there wasabout a seven-fold difference in the apparent Ca2+ affinities: the EC50for the gating effect of Ca2+ on wild-type channels was 175 nM,whereas the Kd for Ca2+ in the C-terminal fragment binding studieswas 1.3 µM. We ascribe these differences to the very different con-texts under which the measurements were conducted. The Kd wasmeasured using a Ca2+-binding domain (hH1-EF) extracted fromthe channel, whereas the EC50 was determined by measuring a volt-age-dependent gating process in a population of channels in theintact cell. Whereas the Ca2+-binding event is coupled to interactionsthat lead to changes in the gated state of the channel, the latter (EC50)measurement incorporates factors beyond the Ca2+ affinity alone.Hence, although the absolute values differ, the parallel changes inCa2+ sensitivity induced by the EF-hand mutations support the pro-posal that the Ca2+ effect can be assigned to the EF-hand domain inthe proximal C terminus.

Recent studies15 have described indirect effects of Ca2+ on multi-ple Na+ channel isoforms through the actions of both CaM and CaMkinase. Inhibition of CaM altered both the voltage-dependent avail-ability and the fast current decay of skeletal muscle Na+ channels15.CaM did not have this effect on hH1, but did inhibit a slowly inacti-vating kinetic component brought about by sustained depolariza-tion14. Inhibition of CaM kinase slowed both the rate of currentdecay and the rate of entry into inactivation, and also induced thevoltage dependence of hH1 availability; notably, all three effects wereindependent of CaM, and did not extend to the skeletal muscle isoform15. Similarly, the direct effects of Ca2+ on hH1 voltage-dependent availability through the EF-hand are CaM independent:the 290–309 CaM-inhibitory peptide did not diminish the

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 3 MARCH 2004 223

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

Ca2+-dependent effects measured on hH1 availability (Fig. 2b).Because 290–309 is a highly specific inhibitor of CaM but not CaMkinase, the use of this peptide should not confound our results byinfluencing CaM kinase regulation.

It remains unclear whether Ca2+ can directly modulate noncardiac Na+ channel isoforms through the C-terminal EF-hand. An earlier report15 found that intracellular addition of10 mM BAPTA eliminates the Ca2+-dependent CaM-inducedhyperpolarizing shift in availability of the skeletal muscle isoform.This finding is not sufficient to exclude the possibility that Ca2+

alone may regulate the skeletal muscle isoform; however, given thatthe Ca2+-dependent CaM effect on the skeletal muscle isoform isdirectionally opposite to the EF-hand-mediated effect we observeon the cardiac isoform, direct Ca2+ regulation of the EF-hand inskeletal muscle Na+ channels may prove difficult to distinguishbecause the gating effects of Ca2+ in skeletal muscle channels maycounterbalance, leading to a more subtle net effect. Numerous single-residue C-terminal sequence changes, both inside and out-side the EF-hand region, markedly influence Na+ channel inactiva-tion gating21,30. These differences could explain the isoformdifferences observed in Ca2+-dependent gating effects. It is increas-ingly clear that several aspects of Na+ channel gating are regulatedby intracellular Ca2+, and that mechanisms mediating these effectsinvolve both direct (EF-hand-mediated) and indirect (CaM14 andCaM kinase15) actions by Ca2+.

L-type Ca2+ channels (Cav1.2) also contain an EF-hand domainalong with an IQ domain in the C terminus13, suggesting functionalsimilarities between those channels and hH1. Although the EF-handand an IQ motif are present in both channel subtypes, Ca2+ channelsoperate by mechanisms that are not fully analogous to their Na+ chan-nel counterparts. For example, Ca2+ binding to the EF-hand does notdirectly cause changes in channel gating in Cav1.2 (ref. 27)Additionally, the IQ motif mediates a high-activity gating mode28 notobserved in hH1. A functional similarity between the two channelsubtypes is that the IQ motif is also the determinant of Ca2+- andcalmodulin-dependent inactivation29. These contrasts between hH1and Cav1.2 show that similar structural motifs may confer diversefunctional effects on voltage-gated ion channel proteins.

Implications for cardiac arrhythmia syndromesOur findings suggest the C-terminal EF-hand domain has a modula-tory role in an inherited form of the long QT syndrome (D1790G;Fig. 3d). Although the putative loci regulating fast and slow inactiva-tion in the Na+ channel were originally localized to the domain III–IVlinker and P-segments, respectively, additional loci throughout thechannel were eventually shown to influence both gating processes.Several inherited mutations in the hH1 C terminus are associated withdefects in either fast or slow inactivation, and the insertion mutant(1795insD) causes defects in both gating processes, causing both thelong QT and Brugada syndromes in the same family21. Notably, boththe IQ and EF-hand motifs in the C terminus are adjacent to many ofthese arrhythmia-related loci. It is possible that inherited mutations inthe C terminus may modulate hH1 inactivation gating by altering thefunction of these two adjacent Ca2+ regulatory elements, either sepa-rately or in combination.

The structural model of the EF-hand domain (Fig. 1c) serves as apoint of reference for generating initial hypotheses to clarify molecu-lar mechanisms that underlie disease-causing mutations in the hH1C terminus. For example, the model suggests that the insertionwithin the Ca2+-binding loop (1795insD) associated with both LQT3and the Brugada arrhythmia syndromes21 will alter the register of

side chains coordinating the Ca2+ ion. E1784K, another LQT3 muta-tion30, is located adjacent to the Ca2+-binding loop, and the modelsuggests that this mutation could disrupt the network of hydrogenbonds critical to the structural stability of the binding loop19.Electrophysiologic, biophysical and structural studies of hH1 con-structs carrying these disease-linked mutations should provideinsight into how modified Ca2+ binding in the EF-hand region influ-ences hH1 dysfunction, and thereby clarify the mechanisms of theseinherited arrhythmia syndromes.

Further analysis is required to understand the relative importanceof the Ca2+ effects mediated through the hH1 EF-hand and theirrelation to mutations causing Na+ channel dysfunction. Ca2+ modu-lation of Na+ channels through additional signaling pathways thatinvolve other second messengers and cofactors, including multiva-lent cations and ATP, must be examined. Experiments in a morephysiologic context, such as isolated myocytes and intact heartpreparations, are also required. Of particular relevance is that intra-cellular free Ca2+ rises in acquired conditions in which the heart isparticularly susceptible to fatal arrhythmias, such as during myocar-dial ischemia. As the maintenance of Na+ channel function seems tobe a crucial determinant of survival under these conditions1, thepotential of the EF-hand as an antiarrhythmic target deserves exten-sive evaluation.

METHODSMutagenesis and transfection. Site-directed mutagenesis was carried out onhH1-α subunit cDNA, and this cDNA was subcloned into the expression vectorpCGI for bicistronic expression of the channel protein and green fluorescentprotein, as described31. Cultured cells (tsA201) were transiently transfectedwith either wild-type or mutant (D1790G or 4×) cDNA (1.5 µg). Green cellswere selected for electrophysiological analysis 24 h later. The cells were main-tained on culture plates using DMEM with FBS (10%) and 1% (v/v) penicillin-streptomycin. In experiments where both the α and β subunits were used,cultured tsA201 cells were cotransfected with a 1:1 molar ratio of the Na+ chan-nel β1 subunit (provided by A. George, Vanderbilt University, Nashville,Tennessee, USA).

Electrophysiology and data analysis. INa was recorded at 22 °C and analyzed(mean ± s.e.m.) as described31. Voltage-clamp protocols are described in thetext or figure legends. A Boltzmann function (y = [1 + exp{(V – V1/2) / k}]–1)was fitted to the availability curves to determine the membrane potential elicit-ing half-maximal inactivation (V1/2). All experiments were done ∼ 6.5 min aftermembrane rupture, to allow full equilibration of the pipette solutions. Dose-response relations were determined by plotting the V1/2 at each Ca2+ concentra-tion, and fitting y = A1 + [(A2 – A1) / (1 + 10(log x0 – x) p)] to the data, where x isthe 50% concentration effect (EC50) and P is a slope factor. Activation curveswere analyzed by fitting to a biexponential function of the form y = A1(1 –exp[–t / τ1]) + A2(1 – exp[–t / τ2]).

The bath solution contained 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2,1 mM MgCl2 and 10 mM HEPES, pH 7.35 with CsOH. The pipette solutionused to mimic Ca2+-free conditions contained: 10 mM NaF, 100 mM CsF,20 mM CsCl, 20 mM BAPTA, 10 mM HEPES, pH 7.35 with CsOH. For theconcentrations of free Ca2+ used in the dose-response analysis, 20 mM BAPTAwas used with 0 CaCl2, 8.94 mM CaCl2, 10.9 mM CaCl2 and 13.4 mM CaCl2 tocreate solutions where the free Ca2+ concentrations were 0, 100, 150 and250 nM, respectively32. For higher (micromolar) Ca2+ concentrations, theBAPTA concentration was reduced to 1 mM to avoid precipitation of thepipette solution. For concentrations of 1 µM and 10 µM free Ca2+, 1 mMBAPTA was used with 0.9 mM Ca2+ and 1.0 mM Ca2+, respectively. The avail-ability curves (Fig. 2) indicate that a substantial portion of the voltage shiftoccurred between 0 and 250 nM, where the BAPTA concentration is fixed (see Fig. 2a legend), suggesting the Ca2+-dependent changes observed do notarise from lowering the BAPTA concentration to achieve high (micromolar)concentration of free Ca2+.

224 VOLUME 11 NUMBER 3 MARCH 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol

A R T I C L E S

Computational analysis. Multiple sequence alignments and analyses were done with AMPS and AMAS33. Threading calculations were done using THREADER 3.3 (ref. 34), which identifies the known three-dimensional structures that best accommodate a given sequence. The library of three-dimensional structural templates (tertiary fold templates) had a representative for all unique three-dimensional protein structures reported in the current Protein Data Bank. The molecular graphics package MOE (Chemical Computing Group;http://www.chemcomp.com) was used for all structure-based sequencealignments and homology modeling (Fig. 1b,c). For the models, amino acidside chains were built onto the consensus backbone (Fig. 1b) using a system-atic conformational search procedure to select optimal side chain rotamers(http://www.chemcomp.com/feature/rotexpl.htm). The starting structurewas refined with conjugate gradient energy minimization and assessed forstructural integrity. PROCHECK35 was used for structure assessment.

Protein expression and purification. A pSV278 vector was constructed forexpression of recombinant hH1-EF (the first 148 residues of the hH1 C-terminal region) fused to an N-terminal His6-tagged maltose-binding pro-tein with a thrombin cleavage site. Standard molecular biology techniques wereused to insert the PCR amplified fragment (forward primer: 5′ CGCGGATCC-GAGAACTTCAGCGTGGCC; reverse primer: 5′ CCGGAATTCCTAAGAGC-GTTGCAGCAGGTG) into the BamHI-EcoRI site of pSV278. The protein wasoverexpressed in Escherichia coli host BL21(DE3) cells (Novagen). Unlabeledprotein was prepared by growing cells in LB medium at 37 °C. Uniform 15Nlabeling for NMR was done by growth in M9 minimal medium containing15NH4Cl and glucose as the sole nitrogen and carbon sources. The protein waspurified by Ni-NTA affinity chromatography (Qiagen), followed by cleavage ofthe His-tag and final purification by anion-exchange chromatography(Amersham Biosciences).

Spectroscopy. Calcium-free hH1-EF protein samples were prepared in twoways: (i) dialysis against 10 mM HEPES, 1 mM BME, 10 mM EDTA, pH 8.0,followed by three further dialysis steps in EDTA-free Chelex-100 treated buffer;(ii) three steps of concentration and resuspension in 10 mM HEPES, 1 mMBME, 1 mM BAPTA, pH 8.0; followed by three steps of concentration andresuspension in BAPTA-free Chelex-100 treated buffer.

CD spectra were recorded at 25 °C over a range of 190–260 nm using a JascoJ-810 spectropolarimeter . The measurements were carried out in 1-mm quartzcuvettes prepared by soaking in EDTA, followed by thorough rinsing withChelex-100-treated H2O.

Gradient-enhanced 1H-15N HSQC NMR spectra were recorded at 27 °C on aBruker spectrometer operating at 600 MHz. The solution contained ∼ 0.1 mM15N-enriched protein in a buffer containing 10 mM HEPES, 1 mM BME, 1 mMCaCl2, pH 8.0, 90% H2O/10% D2O. The NMR spectra were processed and ana-lyzed using XWinNMR (Bruker, Germany).

Steady-state fluorescence emission spectra were recorded at ambient tem-perature using a SPEX FLUOROMAX spectrofluorometer (Spex Instruments)scanning 300–375 nm (λexcit = 280 nm) and 320–375 nm (λexcit = 295 nm),with slit widths set to 5 nm. A 3.2-ml quartz cuvette was prepared by soaking inEDTA followed by thorough rinsing with Chelex-100-treated H2O. Calcium-free protein in a buffer containing 10 mM HEPES and 1 mM BME at pH 7.0was used for titrations. Protein concentration was determined by amino acidanalysis. Binding constants were obtained by fitting to a standard binding equa-tion using CaLigator 1.05 (ref. 36).

ACKNOWLEDGMENTSWe thank S. Stepanovic for invaluable technical assistance, L. Mizoue for providingpSV278 and N. Pokala (University of California Berkeley) for his generous gift ofpSV272. This work was supported by operating grants (to M.E.A., T.P.L., W.J.C.and J.R.B.) from the US National Institutes of Health and a predoctoral fellowshipfrom the American Heart Association.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 13 October 2003; accepted 29 January 2004Published online at http://www.nature.com/natstructmolbiol/

1. Echt, D.S. et al. Mortality and morbidity in patients receiving encainide, flecainide, orplacebo. N. Engl. J. Med. 324, 781–788 (1991).

2. Bennett, P.B., Yazawa, K., Naomasa, M. & George, A.L. Molecular mechanism for aninherited cardiac arrhythmia. Nature 376, 683–685 (1995).

3. Chen, Q. et al. Genetic basis and molecular mechanism for idiopathic ventricular fibrilla-tion. Nature 392, 293–296 (1998).

4. Dumaine, R. et al. Multiple mechanisms of Na+ channel-linked long-QT syndrome. Circ.Res. 78, 916–924 (1996).

5. Wang, D.W., Yazawa, K., George, A.L. & Bennett, P.B. Characterization of human cardiacNa+ channel mutations in the congenital long QT syndrome. Proc. Natl. Acad. Sci. 93,13200–13205 (1996).

6. Bezzina, C. et al. A single Na(+) channel mutation causing both long-QT and Brugadasyndromes. Circ. Res. 85, 1206–1213 (1999).

7. Benhorin, J. et al. Identification of a new SCN5A mutation, D1840G, associated with thelong QT syndrome. Mutations in brief no. 153. Online. Hum. Mutat. 12, 72 (1998).

8. An, R.H. et al. Novel LQT-3 mutation affects Na+ channel activity through interactionsbetween α- and β1-subunits. Circ. Res. 83, 141–146 (1998).

9. Wehrens, X.H., Abriel, H., Cabo, C., Benhorin, J. & Kass, R.S. Arrhythmogenic mecha-nism of an LQT-3 mutation of the human heart Na(+) channel α-subunit: a computa-tional analysis. Circulation 102, 584–590 (2000).

10. Cormier, J.W., Rivolta, I., Tateyama, M., Yang, A.-S. & Kass, R.S. Secondary structure ofthe human cardiac Na+ channel C terminus: evidence for a role of helical structures inmodulation of channel inactivation. J. Biol. Chem. 277, 9233–9241 (2002).

11. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignmentsearch tool. J. Mol. Biol. 215, 403–410 (1990).

12. Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acids Res. 25, 3389–3402 (1997).

13. Peterson, B.Z. et al. Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels. Biophys. J. 78, 1906–1920 (2000).

14. Tan, H.L. et al. A calcium sensor in the sodium channel modulates cardiac excitability.Nature 415, 442–447 (2002).

15. Deschenes, I. et al. Isoform-specific modulation of voltage-gated Na(+) channels bycalmodulin. Circ. Res. 90, E49–E57 (2002).

16. Lewit-Bentley, A. & Rety, S. EF-hand calcium-binding proteins. Curr. Opin. Struct. Biol.10, 637–643 (2000).

17. Shaw, G.S., Hodges, R.S. & Sykes, B.D. Calcium-induced peptide association to form anintact protein domain: 1H NMR structural evidence. Science 249, 280–283 (1990).

18. Linse, S. & Forsen, S. Determinants that govern high-affinity calcium binding. Adv.Second Messenger Phosphoprotein Res. 30, 89–151 (1995).

19. Strynadka, N.C. & James, M.N. Crystal structures of the helix-loop-helix calcium-bindingproteins. Annu. Rev. Biochem. 58, 951–998 (1989).

20. Shaw, R.M. & Rudy, Y. Electrophysiologic effects of acute myocardial ischemia. A mech-anistic investigation of action potential conduction and conduction failure. Circ. Res. 80,124–138. (1997).

21. Veldkamp, M.W. et al. Two distinct congenital arrhythmias evoked by a multidysfunc-tional Na(+) channel. Circ. Res. 86, E91–E97 (2000).

22. Tsien, R.Y. New calcium indicators and buffers with high selectivity against magnesiumand protons: design, synthesis, and properties of prototype structures. Biochemistry 19,2396–2404. (1980).

23. Christova, P., Cox, J.A. & Craescu, C.T. Ion-induced conformational and stability changesin Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a moltenglobule. Proteins 40, 177–184 (2000).

24. Veeraraghavan, S. et al. Structural independence of the two EF-hand domains of cal-tractin. J. Biol. Chem. 277, 28564–28571 (2002).

25. Theret, I., Baladi, S., Cox, J.A., Sakamoto, H. & Craescu, C.T. Sequential calcium bindingto the regulatory domain of calcium vector protein reveals functional asymmetry and anovel mode of structural rearrangement. Biochemistry 39, 7920–7926 (2000).

26. Tateyama, M., Rivolta, I., Clancy, C.E. & Kass, R.S. Modulation of cardiac sodium chan-nel gating by protein kinase A can be altered by disease-linked mutation. J. Biol. Chem.18, 18 (2003).

27. Zhou, J. et al. Feedback inhibition of Ca2+ channels by Ca2+ depends on a shortsequence of the C terminus that does not include the Ca2+-binding function of a motifwith similarity to Ca2+-binding domains. Proc. Natl. Acad. Sci. USA 94, 2301-2305.(1997).

28. Wu, Y., Dzhura, I., Colbran, R.J. & Anderson, M.E. Calmodulin kinase and a calmodulin-binding ‘IQ’ domain facilitate L-type Ca2+ current in rabbit ventricular myocytes by acommon mechanism. J. Physiol. 535, 679–687 (2001).

29. Zuhlke, R.D., Pitt, G.S., Deisseroth, K., Tsien, R.W. & Reuter, H. Calmodulin supportsboth inactivation and facilitation of L-type calcium channels. Nature 399, 159–162(1999).

30. Wei, J. et al. Congenital long-QT syndrome caused by a novel mutation in a conservedacidic domain of the cardiac Na+ channel. Circulation 99, 3165–3171 (1999).

31. Tan, H.L. et al. A sodium channel mutation causes isolated cardiac conduction disease.Nature 409, 1043–1047 (2001).

32. Bers, D.M., Patton, C.W. & Nuccitelli, R. A practical guide to the preparation of Ca2+buffers. Methods Cell Biol. 40, 3–29 (1994).

33. Livingstone, C.D. & Barton, G.J. Protein sequence alignments: a strategy for the hierar-chical analysis of residue conservation. Comput. Appl. Biosci. 9, 745–756. (1993).

34. Jones, D.T., Taylor, W.R. & Thornton, J.M. A new approach to protein fold recognition.Nature 358, 86–89 (1992).

35. Laskowski, R, MacArthur, M.W., Moss, D.S & Thornton, J.M. PROCHECK: a program tocheck the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291(1993).

36. Andre, I. & Linse, S. Measurement of Ca2+-binding constants of proteins and presenta-tion of the CaLigator software. Anal. Biochem. 305, 195–205 (2002).

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 11 NUMBER 3 MARCH 2004 225

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

stru

ctm

olbi

ol