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CHAPTER 5 Sarcolemmal Ca Channels

Noncommercial, Private Translation by Algebra only for Personal Understanding of Cardiac Physiology. Distribution is Prohibited. , , .

121

CHAPTER 5

Ca INFLUX via SARCOLEMMAL Ca CHANNELS

Ringer(1883) [Ca] .

Ca currents slow inward current ( ) Isi ,

(peak current) ms .353 (patch-clamp)

(isolated myocyte technique) Ca (ICa) ~2-3 ms

(peak value) ( 50). , slow current

. , INa ICa

( 45). action potential inward ICa AP plateau ,

(contraction) / .

Ca CHANNEL TYPES

Hagiwara (1975) (single cell type) Ca (class)

. Nowycky (1985) dorsal root ganglion Ca ,

(nomenclature) . L-type Ca channels (Large

conductance; 110 mM Ba ~25 pS), (Long lasting openings;

Ba ), 1,4-dihydropyridines (DHPs) (sensitivity)

(Larger depolarization) (activation) . T-type channels

(Tiny conductance; 110 mM Ba ~8 pS), (Transient openings), DHP

(), (more negative Em) . N-tpye Ca

channels T L (Neither), (Neuron) (predominantly) ,

(intermediate) .

Ca current type (; P/Q,

R-type) , (neuroendocrine) .

T-type Ca (low-voltage-activated (LVA) type), type

high-volated-activated (HVA) (, -Em-dependence-

). 15 10 Ca354

353 ) Rougier , 1969; Mascher Peper, 1969; Beeler Reuter, 1970; Ochi, 1970 354 ) Em-dependent Ca channel genes



122

(properites) alternate names .

L, T, N , L-type

. , -conotoxin L-type Ca ,

L-typer Ca (McCleskey , 1987). , L-type

activation inactivation kinetics ~10 (Bean, 1989).

L-, T-type Ca (, 1C, 1D, 1G, 1H).

L-, T-type Ca (; 50 16), N-type (Bean,

1985, 1989; Nilius , 1985). ICa,L , cardiac ICa,T

( 50B, Hirano , 1989). Test Em 20 mV ICa,L ICa,T (

50B), 0 mV peak ICa.L ICa,T 3 . L-type Ca

(dominant) , T-type ICa . 50C 4

(total) ICa - (current-voltage relationship) . Netagive Em( 40 mV)

(hump) ICa,T (relative amount) .

, pacemaker (Hagiwara , 1998; Bean,

1989). , ICa,T (modest)(Mitra Morad, 1986)

355, (calf), , , .356 (neonatal

rat ventriclular myocytes) ICa,T (Wetzel , 1993; Gaughan , 1998), (Martinez , 1999)

(Nuss Houser, 1993) (ventricular hypertrophy) ICa,T

(reappear).

355 ) bullfrog: . 356 ) Bean, 1989; Nuss Houser, 1993; Yuan Bers, 1994; Yuan , 1996



123

, T-type Ca , , (



124

) .357 ICa,T pacemaker conduction cells

, pacemaker Em , ICa,T

pacemaking , (112, Hagiwara, 1988; Wu

Lipsius, 1990). ICa,T inactivate , ICa,T Ca

flux ICa,L ,

. . , ICa,L pacemaking SR

Ca release (trigger) SR Ca store (refill) .

ICa,L inactivation Ca358 ICa,L (transient) T-type ICa ICa,L kinetics

. , L-type T-type Ca Ba charge carrier

( L-tpye inacitvation ; ; 50A). T-type

Em-dependence ( 50). ICa,T (substantial) -

(holding Em 80 90 mV) negative Em (shoulder) .

T-, L-type ICa , N-, P/Q- R-currents

.

ICa,T T-tubule(T-)( 1)

.359 T-type Ca T-tubule . , L-type Ca

(T-tubule) (1 ).360 ,

, (cardiac myocytes) Ca channels ICa L-type Ca channels ICa,L

.

MOLECULAR CHARACTERIZATION OF Ca CHANNELS

L-type Ca dihydropyridines (DHPs) . DHPs Ca

blockers antagonists361 (; nifedipine, nisoldipine, nitrendipine, isradipine),

DHPs Ca agonists (; Bay K 8644 (-)enantiomer). Agonist

(agents) open time() (prolong) ( 61). ,

L-type Ca DHP, T- DHP

L-type , .362

357 ) Thus T-type current is typically small or absent in ventricular myocytes, but may be more prominent during development and hypertrophy. ) , may, can . , (evidences) .

358 ) Kokubun Irisawa, 1984; Lee , 1985; 137 . 359 ) The cardiac cell types in which ICa,T is more prominent also happen to be the cells which have less

extensive T-tubules. 360 ) , but L-type Ca channels in cardiac and skeletal muscle may be concentrated there. 361 ) agosnist antagonist 362 ) Curtis Catterall, 1984; Borsotto , 1984; Flockerzi , 1986



125

Ca 5 (1, 2, , , ) , .363

1(1S) clone (1C, Mikami , 1989)

. 1 Ca

. , 1 , DHP, (phenylalkylamine),

(benzodiazepine) (receptors)364, protain kinase A (PKA), CaMKII365, protein kinase C (PKC)

(OCallahan , 1988) (sites) . 1C (coding) DNA

(lacking) transfection Em-dependent ICa (Perez-

Reyes , 1989). , 1 Em-dependent Na tetrameric(4) K(

38) . , 1C 4 (I-IV), transmembrane span

6 (S1-S6, S4 -charged), S5 S6 pore , C-terminal

. 1C alternate splicing variants (Bers Perez-Reyes, 1999 ). , 1D

(Takimoto , 1997; Wyatt , 1997), 1C 1D

.

4 (Em-dependent channels) , Ca

S4 span, 10-15 a.a. (stretch) 3 a.a. . ,

363 ) Tanabe , 1987; Ellis , 1988; Ruth , 1989; Jay , 1990; Bosse , 1990 364 ) Galizzi , 1986; Sharp , 1987; Sieber , 1987; Vaghy , 1987; Striessnig , 1990a,b 365 ) Curtis Catterall, 1985; Hosey , 1986, 1987; Imagawa , 1987a; Nastainczyk , 1987



126

Ca Em-dependent gating . Ca (activation)

. , Ca(1S) 1C . Tanabe (1991)

(chimeric) Ca , 1S 1C I

. , Spaetgens Zamponi (1999) II III (

IV) 1C 1E Em inactivation .

Ellis (1988) 2 , 2

2 gene (Jay , 1991). , 2 (cleaved)

2 . 2

(disulfied link) , ( 51).

2 , alternative splicing splice variants

.366 2a , 2b , 2c, 2d

( ). Klugbauer (1999) 2 isoform

.

2 1C , DHPR binding site (expression), gating currents, ionic

currents ~2 .367 2

. , 2 DHP

(apparent) 4 (Wei , 1995) opening closing (Bangalore ,

1996; Felix, 1999 ). 2 (anticonvulsant) gabapentin drug

(Gee , 1996), theraputic impact .

2 1 III

. 2 (

transmembrane spanning )368(Gurnett , 1997; Felix , 1997).

Ca (Ruth , 1989; Jay , 1990). , 1S

DHP binding , (allosteric

action) (alter) (Suh-Kim , 1996). ,

brain (additional) (Letts , 1998).

4 (1-4), isoform 2 . 2 (exons)

alternative splicing N-terminus 1

(Perez-Reyes Schneider, 1995; De Waard , 1994). 2a isoform

(Qin , 1998). 3 1 variants (detected)(Collin , 1993;

366 ) Williams , 1992; Kim , 1992; Angelotti Hoffman, 1996 367 ) Wei , 1995; Singer , 1991; Bangalore 368 ) Most of the component seems merely to anchor the 2 subunit in the membrane (since other

transmembrane spanning domains can substitute), whereas the heavily glycosylated extracellular 2 domain seems to interact with the 1 subunit, at least in domain III.



127

Hullin , 1992). Northern analysis 2 isoform , 3

. 2 (antibody) DHP binding sites

80% (immunoprecipitation) . , 2 cardiac .

2 1C , 10 , inactivation kinetics

, steady-state inactivation (shift), high affinity DHP binding sites369 (number)

.370 2 -adrenergic- ICa (Haase ,

1993; Bnemann , 1999). (moleular chaperone)

nascent 1C (folding) (Gao

, 1999). , pore 371 1C

DHP (Mitterdorfer , 1994, 1998).

1 (specific) AD BID

51 (De Waard , 1994; Pragnell , 1994). 1

(site)(AID) I-II loop. loop splice variants , 1 variants

. 1

(BID) 2 highly spliced region , 1- functional

variants . , splice variants N-terminus(2a vs. 2b)

splice variants 1E channel kinetics (Olcese ,

1994; Qin , 1996). 1a 1b 2a palytoylated .

Palmytoylated residue (Qin , 1998;

Chien , 1996).

N-, P-type Ca 1A 1B AID ,

G-protein (modulated).372 (various) 7 transmembrane

spanning receptors373 heterotrimeic G-protein . G

Ca 1 I-II loop ( Ca channel

) ICa (rate) (amplitude) (depression). ICa

(depressant effect)374 , ICa Em-dependent

facilitation. (firing at higher frequencies) Ca influx

369 ) DHP binding sites. , emzyme drug / kinetics RyR, DHPR binding sites low affinity bnidnig sites high affinity binding sites . high affinity binding sites.

370 ) Perez-Reyes , 1992; Neely , 1993; Mitterdorfer , 1994 371 ) Costanin , 1998; Yamaguchi , 1998; Gerster , 1999 372 ) Dolphin, 1998; Ikeda Dunlap, 1999 . 373 ) G-protein coupled receptors (GPCR) . 374 ) (This depressant effect on ICa) which may be tonic



128

. G Ca 1 (inhibition) .

G-protein ( Em-dependent relief) ICa

, Ca .

51 1 ( ) cardiac ryanodine

receptor (II-III loop carboxy tail), Carboxy tail

(Ser-1928), EF-hand IQ motifs(Ca- inactivation), 3 Ca



129

blockers(DHPs, , ) (IIIS5, IIIS6, IVS6) . 1C,

2, 2 200, 175, 60 kDa(2- 150 2, 30 ).

3 T-type Ca 1G, 1H, 1I .375 376

L-type Ca (; I-IV , S1-S6 transmembrane spans pore loop).

S4 pore (region) . , T-type high voltage Ca

sequence identity (< 15%). , III pore loop

EEEE locus Ca glutamate aspartate

(137 ). , T-type Ca SKD , 1C TFE

. 1C 1H (motif) 1) (I-II region) 2) Ca

inactvation EF hand (putative) ( ).

() , ICa,T Ca- inactivation . Mebefradil

ICa,T (inhibitor), T-type Ki ~1 uM(ICa,L

block 11 , Cribbs , 1998). Ni ICa,L block, 1H

( 15), 1G 1I Ni- ICa,L (Lee , 1999b). pacemaker

ICa,T Ni- , 1H . ,

ICa,T Ni- , 1G .

1G 1H mRNA . 1H 377

, mRNA (Cribbs , 1998).

T- (subtype) pacemaker ,

. ICa,T 1 ,

ICa,T ( Ca) .

1E ICa,T (Peidras-Rentera , 1997),

ICa .

Ca CHANNEL SELECTIVITY AND PERMEATION

McCleskey Almers (1985) Ca pore ~6 (

-tetraalkylammooium- ). , Ca size exclusion

(selectivity) . Hagiwara (1974) Ca Ba

Ca , Ba Co block . , Co Ba

binding site Ca Ba ,

binding site . Ba

375 ) expressed in vivo . 376 ) Perez-Reyes , 1998; Cribbs , 1998; Lee , 1999a,b 377 ) cDNA library



130

vs. Ca . Ba (does not stick)

, .

Hess (1986) (asymmetrical salt solution) reversl potential

Ca ( 17).

(permeant ions) single channel conductance . 2 , Ca

1 (INS, inward current). Na pore Ca

. , [Ca]o Ca Ca

block378, Kd ~1 uM [Ca] ( 52A).379 Mg INS block(Ki ~50 uM),

unblocked INS Mg Ca chelate (, EGTA

EDTA ). 52A [Ca]o , ICa [Ca]o mM

, Kd ~14 mM (saturated)(Hess , 1986). (disparity)

Ca Ca binding site .

Anomalous mole fraction effect (ANFE) ,

site (Yue Marbn, 1987; Friel Tsien, 1989). Ca

Ca Ba ( [Ca]+[Ba])

(Hess Tsien, 1984).

Almers McCleskey(1984), Hess Tsien(1984) permeation

Ca 2 Ca channel permeation model ( 52C).

(unoccupied) Ca , Na, Ca (potential energy; G/RT)

(Ca) (Na) . (well; ) 2 Ca

binding sites. Ca site prodile (2 ). Ca

378 ) Indeed, as [Ca]o is increased Na current through cardiac and skeletal Ca channels is blocked by Ca.. 379 ) Almers McCleskey, 1984; Almers , 1984; Hess Tsien, 1984



131

sites pore . Na open site ,

(dwell) . , Na uM

[Ca] block. Ca (single occupancy by Ca)

sites Ca Kd ~0.5 uM ! ~10 mM . , Ca site

(repulsive force) , Ca 50% pore (Em = 0

). (inward directed) driving force Ca entry (ECa

~+120 mV), inward Ca current . , double occupancy(

) (off-rate380)

, 2 Ca flux

. , Ca flux double occupancy mM [Ca]

( 52B). Pore binding sites

(Yue Marban. 1990).

, (strict sequential model) (heuristic)

, Ca

(Armstrong Neyton, 1992; Dang McCleskey, 1998). , (selectrivity

filter) glutamate , EEEE locus pore loop

(EI, EII, EIII, EIV, Yang, 1993).

EGTA Ca . (glutamates)

380 ) pore ()



132

(glutamines) 1C (heterologically)

(Yang, 1993; Ellinor , 1995), Li INS 50% block [Ca]o

( 53A). ,

Ca block ( 1000). (point mutation)

Kd(Ca) , .381 EEEE locus

382 . , 53B EEEE

locus pore Ca (Veradi , 1999).

Ca , Ca

(Kd ~1 uM) Ca (Kd ~10 mM; Yang , 1993;

McCleskey, 1999) . ,

two-sites model . ,

Ca , Ca ( )

Ca ( 53C-E). , Ca Ca ( Na

flux block) Na .

381 ) Since single point mutation alter Kd(Ca) there cannot be 2 discrete independent sites. 382 ) Ellinore , 1995; Chen , 1996a; Chen Tsien, 1997



133

()

, Nonner Eisenberg (1998) (rigid electronegative tube)

Ca continuinuous Poisson-Nernst-Planck(PNP) model .

, Ca Ca Na block . EEEE

locus (McCleskey, 1999),

.

Ca (protons)(Prodhom , 1987) (multavalent cations)

(Lansman , 1986) flickering block . blocking events

blocking event (unresolvable) , conductanve

. pH INS , pH 9 full conductance 85 pS .

flickering block pore (transient)

, () on-, off-rates .

Ca block .

block, . voltage-dependence for the relief of block

, ( negative Em) (forced)

(Lansman , 1986).

, Mg Ca , Ca (impermeable)

.383 , 1S, 1C, 1D 4 P-loop cores (identical)

. , Ca P-loop

(Yuan , 1996) , core P-loop

. (symmetrical solutions) Ca - (lenear

relationship)(Rosenberg , 1986), (permeation pathway)

.

, Ca current conductance

. , conductance (relatively

linear relationship) . (in the limit)

, pore . , access

resistance , ()

(Peskoff Bers, 1988; Bers Peskoff, 1991). pore

(ion depletion) (accumulation) (local

potential) pore (slightly) .384 , Ca

[Ca]o (local negative potential)

383 ) Almers Palade, 1981; Hess, 1986 384 ) This effect is slightly mitigated by the local potentials created by the ion depletion and accumulation and

any fixed negative charges on or near the channel.



134

(mediun) Ca (mouth) . flux

2rD[X]o , X , r (; 0.3 nm), D

(Na Ca ~10-5 cm2/s, ~310-6 cm2/s). narrow Na, K , 140 mM

~50 pA . Ca [Ca]o 2 mM, 110 mM

0.4 pA, 24 pA (Peskoff Bers, 1988). Ca (single

channel) ICa 0.2 pA ,

Ca depletion (Bers Peskoff, 1991; 8 118).

, [Ca]o Ca depletion/accumulation ICa

. channel conductance ICa

( 54A ). [Ba]o Conductance()

depletion [Ba]o (saturate). 54B-C McDonald

(1994) 24 (13 , 11 ) (compile)

Ba, Ca conductance . Ca, Ba conductance 10 pS 27 pS, 4 mM



135

[Ca]o 10 mM [Ba]o Km( ) . Km

( 53E) . ,

Na conductance Ca 10 (charge ion

20). , Ca ( Ba) flux ,

binding site(s) Ca dissociation rate().

Ca flux Ca current . block

, ICa Ca

. , PCa/PCs >1000 . 54D

Goldman-Hodgkin-Katz(GHK) Ca Cs .

;

[ ] [ ] ( )( )( )

[ ] [ ] ( )( )( )

+

m

moiCs

m

mioCamCa kE

kECsCsPkE

kECaCa4PFkE=Iexp1

exp2exp1

2exp (1)

k = 1/(25.7 mV), PCa/PCs = 1000 . Ca flux, Cs flux. +10

mV ICa flux Ca influx (Ca influx 4%

). , reversal potential (Erev= +52 mV) ICa 0 Ca influx +10

mV 11% . Erev Ca influx Cs efflux

Em. (net) Ca influx Ca equilibrium potential (ECa = +125 mV)

. Zhou Bers(2000) Ca entey Erev Em ICa

(demonstrated)( Ca channel influx -fluorescent indicator- ICa

-voltage clamp- ). GHK

, GHK . pore binding

sites, (ion-ion interaction) .385 ICa , Erev

Ca influx . , Ca influx

Erev (; E-C coupling ).386

NUMBERS OF Ca CHANNELS

Schwartz (1985) , (functional) L-type

DHP (specific DHPRs) 30-50 . , L-

type Ca 3-5/um2 (-single channel- -whol cell- ICa

, McDonald , 1986; Tsien , 1983). Bers Shiffel (1993) adult() , ,

385 ) This is due in part to the biding sites in the pore and the ion-ion interactions which are known to exist. 386 ) Ca influx above the Erev for ICa is important to keep in mind experimentally, since..



136

, specific DHP binding , ~150 fmol/mg protein

.387 (homogenate) ,

(~90 fmol/mg protein). 120 mg/cm3 protein, 25%

(extracellular space), (surface-volume ratio) 0.6/um( 1, T- )

, DHPR ~20 DHPRs/um2 . Ca

gating (represent) non-linear charge movement Ca

(3.7-5.5 nC/uF).388 6 elememtary charge (membrane

field) , gating currents 37-57 /um2 .

Ca (N) N = ICa/(iCa po) whole cell current single cell current

. open channel probability(po)389 .

po Ca (true mean open

probability) . McDonald (1986) whole cell ICa po(~0.8)

, BayK8644 Po390 ICa

(Hess , 1984a; Bean Rios, 1989). Lew

(1991) peak current Po 0.03 (inactive mode )

iCa ICa N 18 channels/um2 ,

387 ) Green , 1985; Kokubun , 1986 . 388 ) Field , 1988; Bean Rios, 1989; Hadley Lederer, 1989 389 ) (single channel) . (5-10 30-40) trace (, 50% ) .

390 ) po Po (single channel ).



137

DHPR (13-15/um2) . , Ca DHPR (~15/um2

~300000/cell), peak ICa ~3% Ca . , cardiac DHPR L-type Ca

(discrepancy) . whole cell ICa peak Po 0.03391,

Ca . , Ca , SR

junctions .

Ca CHANNEL GATING

Surface potential and activation

Cardiac ICa , Em ~2-7 ms peak

( 45 50). Ca Em , voltage

sensitive channels392 surface potential ( ) (; Wilson ,

1983; Hille, 1992). Surface potential (McLaughlin, 1977,

1989).393 55 surface potential (o i) (membrane; m)

, .394

(physiologica medium; 2mM Ca, ~150 mM ionic strength) ICa (Em

= 40 mV) , Gouy-Chapman theory of the diffuse double layer

(Grahame, 1947). Surface potential (arbitary) , ~1 elementary

charge/ 100-300 2 surface charge densities( surface density

) . Kass Krafte(1987) 2

Ca gating (shift)

(, 1/2502). (inner leaflet) , 2

i (Post , 1988). 2

surface charge (screening) , 2

, ,

(McLaughlin , 1981; Bers , 1985). , 1 mM [Ca]o surface [Ca]o [Na]o 25

mM, 700 mM ( surface [Cl] ).

, 55 Em = 40 mV

(trans-bilayer potential) m = 60 mV . 2

surface potential(o) (abolished), Em = 40 mV trans-

bilayer potential (polarize) (m = 88 mV).

391 ) , Schwartz (1985) 1.0 . 392 ) (voltage) (sensitive) (channels) 393 ) The surface potential arises from fixed negative charges on the membrane surface. 394 ) Fig 55 inllustrates the effect that changing surface potential can have on the electric field within the

membrnae, which may be what the voltage sensors of the channnel respond to.



138

trans-bilayer Em

. 50A 115 mM Ba C-V 395 50B 2 mM Ca

Em (rightward shift) ( 2

INS ICa 20 mV ). Ba Ca (activation) (shift)

, Ba Ca ( 2

). Em-dependent channels , Em-dependent

inactivation . 2

(increased excitability) . , [Ca]o

activation/inactivation (negative) Em (shift).

Surface potential effect Debye ( 1 Debye length = ~ 1 nm)

(decay).396 Surface potential Ca gating , Ca

Ca conductance . , Coronado

Affolter(1986) single channel conductance surface potential

(releatively insensitive) (demonstrated). , gating sensor ,

(permeation pathway) o (

pore 2nm

).

395 ) , current-voltage relationship C-V relationship . 396 ) The effect of the surface potential decys exponentially over a few Debye lengths.



139

4 , Em-dependent Ca channels( Ca )

(closed states) , ! (trsnsition) Em-

independent. 40B , Ca gating charge mevement Em- ICa

(negative) Em (Bean Rios, 1989).397

ICa inactivation

Ca inactivation -, Em-(-), [Ca]i-.398 56A INS

inactivation (t1/2 > 500 ms) , test Em(30mV) purely Em-

dependent inactivation .399 , 30 mV

Em 2 (membrane field) m [Ca]o Em = 10mV

0 mV (comparable)( Ca surface charge

). Pulse 0 mV 2 , Em

40-50 mV (positive) , INS inactivation (, Em- inactivation

, Em positive inactivation ).400 56A Ba

(IBa) INS (readily) inactivation(t1/2 = 161 ms), Ba Ca-

(dependent) inactivation (modest) .(Ferreira , 1997). Ca

(charge carrier) , inactivation (t1/2 = 37 ms).

Ca Ca- inactivation . EGTA Ca buffer

Ca local [Ca]i . BAPTA Ca

buffer Ca- inactivation (slow) . E-C

coupling SR Ca L-type Ca local [Ca]i . 56A

ICa trace Ca transient perforated patch mode . ICa

(t1/2 = 17 ms) inactivate (setting) SR Ca ICa inactivation

(major role) . , action potential clamp( 60), normal SR Ca release

ICa,L Ca influx (integration) 50% (Puglisi , 1999).

56B Em 500 ms inactivation

(Hadley Hume), 1987. INS +60 mV 500 ms inactivation

397 ) .., most of the Em-dependence in Ca channel activation is between closed states, with the final closed to open transition being relatively Em-independent. Analogous to Fig 40B, the Em-dependence of Ca channel gating charge movement occurs at more negative Em-than of ICa.

398 ) Lee , 1985; Kass Sanguinetti, 1984; Hadley Hume, 1987 399 ) Fig 56A shows that inactivation of INS is very slow and probably reflects purely Em-dependent inactivation

is very slow in this test Em. 400 ) The divalent cation currents were recorded during pulses to 0 mV. I should note that when Em is 40-50

mV more positive to this range the insctivation of INS increases(i.e. Em-dependent inactivation is more prominent, but still incomplete even at large positive Em).



140

. (strong) positive Em(Ca 401 ) , ICa-, INS

inactivation ( , -purely- -Em-dependent- ).

(intermediate) Em ICa inactivation , inward ICa amplitude

Em-dependence(; ~0 mV . 50C). Ca- inactivation AP(

56A voltage clamp pulse) time scale (overshwhelmingly dominant) inactivation.

ICa Ca- inactivation [Ca]i , Ca entry

(negative feedback control) . Cai- inactivation

, EGTA BAPTA

. , ICa Ca [Ca]i

( ) inactivation . Hfer

(1997) single channel Ca currents , 4 uM [Ca]i Ki .

de Leon (1995) 1C

putative Ca-binding EF-hand region( Ca 1E , Ca-

inactivation ) . 1C EF-hand 1E

, Ca Ca- inactivation . , 1C

Ca-binding site (mutagenesis) (disrupt)

401 ) where little Ca enters



141

(Zhou , 1997). , (calmodulin; CaM) 1C

Ca- inactiation , .402 CaM resting state

Ca . Ca 1 Ca

(Kd = 29 nM) CaM-1C(1C C-terminal IQ motif . 51 1624-5)

. local [Ca] , Ca CaM(4 binding site

) Ca IQ motif inactivation.

, Ca-CaM-IQ motif N-type inacivation

ball and chain .

voltage clamp pulse( action potential) ICa cytosolic Ca transient

inactivation, [Ca]i (recover)

(Sipido , 1995a). (long) action potential L-type Ca (reactivation)

(arrhythmogenic) early afterdepolarizations (EADs) .

57A ICa steady state activaoin availability curve403

( EGTA SR Ca load transients ) (Yuan , 1996).

Inactivation variable() 45mV 15mV 0 .

ICa Em Em inactivate , Ca-

inactivation , (larger) windows current . macimal

steady state window ICa 28mV , conductance

1%, 5%(d ). Em 45mV 15 mV

Ca . Josephson (1984)

window current . Cohen Lederer(1988) window ICa

, SR Ca release , SR Ca

release (maturation)404 cross-signaling .

Ca inactivation, recovery [Ca]i-, Em-. 57B Em 90mV

50mV recovery . Ca recovery AP

. 50mV inactivated frequency-

dependent accumulation405 (; 1Hz). , H-

89, KN-62, KN-93 protein kinase inhibior() 90mV recovery (slow) ,

Em 50mV (resemble)(Yuan Bers, 1994; Li , 1997b). ,

402 ) Zulkhe Reuter, 1998; Peterson, 1999; Qin, 1999; Zulkhe, 1999 403 ) 4 . 404 ) SR

Ca handling L-type Ca channel RyR . 405 ) (-frequeny dependent-) , recovery (accumulation) pool .



142

apparent ICa protocol dependent way .

Ca-dependent ICa facilitation or ICa staircase

Holding potential 40mV , voltage clamp (frequency) ICa

amplitude (pulse-dependent progressive decline)(Tseng, 1988; Hryshko Bers, 1990).

ICa (negative staircase) Ca pulse inactiated state(Em = 40mV)

.

holding potential(80mV), 58 pulse ICa amplitude

(pulse-dependent progrssive increse), inactivation (prominent slowing of

inactivation).406 ICa (positive staircase) Ca-(Ba charge

carrier ), SR Ca . 3

CaMKII-dependent phosphorylation (CaMKII ) Ca-dependent ICa facilitation407

.408 , SR Ca(ICa local Ca flux )

, (Delgado , 1999). Ca-dependent

facilitation 10 mM EGTA (, 20 mM BAPTA

), CaMKII Ca- L-type Ca

.(Hryshko Bers, 1990). ICa Ca entry facilitation effect( )

406 ) Mitra Morad, 1986; Lee, 1987; Argibay , 1988; Boyett Fedida, 1988; Fedida , 1988a,b; Gurney , 1989; Hryshko Bers, 1990; Tseng, 1988; Zygmunt Maylie, 1990

407 ) Ca Ca . 408 ) Yuan Bers, 1994; Anderson , 1994a; Xiao , 1994a; Dzhura , 2000



143

Cai- inactivation .409 , [Ca]i unitary ICa biphasic

effects410 (Hirano Hiraoka, 1994).

Zuhlke (1999) IQ isoleucine (point mutation) Ca-CaM-

inactivation , Ile! Ala inactivation (enhance) Ile!

Glu (abolish). , CaMKII ICa CaM Ca-

inactivation .411 [Bers DM] positive ICa staircase( 58)

ICa inactivation CaM inactivation target CaMKII

(functional shift) . Ca- facilitation

(impact) . , Ca- inactivation

(offset) , Ca (memory

effect) .

ICa voltage-dependent() facilitation chromaffin cells412(Artelejo , 1990, 1992),

(Sculptoreanu , 1993a), cardiac Ca413 . Cardiac Ca

cAMP-dependent protein kinase (PKA) , Ca

(may require)(, G-protein Ca modulation-124

- ). , facilitation positive potential (; > +100 mV) pulse

, (extremely) . ,

+100 mV 60mV 10ms facilitation (,

Em ). , voltage-mediated ICa facilitation

.

AMOUNT OF Ca ENTRY via Ca CHANNELS

ICa Ca (myofilament) Ca

. 0mV square pulse() (voltage-clamp)414 ICa

(waveform) ~10 umol/L cytosol (63; peak ICa = 1 nA,

409 ) This facilitatory effect of Ca entry on subsequent ICa is distinct form, but coexists with the Cai-dependent inactivation discussed above.

410 ) (facilitation) (inhibition) . , .

411 ) Thus the CaM involved in activating CaMKII and ICa may be the same tethered CaM which is involved in Ca-dependent inactivation.

412 ) (Cr) . adrenal medullary cells( ). 413 ) Pietrobon Hess, 1990; Sculptoreanu , 1993b; Xiao , 1994a; Kamp , 2000 414 ) . action potential .



144

120 ms 30 pL cytosol 415). , AP Ca influx

square pulse . 59 square pulse AP

command potential (, aP clamp)416 .417 AP clamp

peak ICa , square pulse . , AP peak(+50mV) Ca

, Em ICa reversal potential (~+60mV) Ca

driving force . Em inactivation driving forve

AP (larger current) . square pulse AP

415 ) LmolLmolH

molCaC

molA /...3.1010301

21

965001

2sec12.010

12

29

=

++

416 ) driving force . , ready resting potential, (stimulation) command potential .

417 ) Doerr , 1990; Arreola , 1991; Yuan , 1996; Grantham Cannell, 1996; Linz Meyer, 1998



145

ICa . 59 Ca influx running integral .

200 ms square pulse Ca influx (Yuan

, 1996). ICa activation/ inactivation . , AP

AP Ca influx (21 vs. 14 umol/L

cytosol).

59 data Ca influx , EGTA (dialyzed)

(, ICa Ca-dependent inactivation ). SR

Ca release (INa/Ca ICl(Ca) ) block

25C 35C AP clamp ICa (Puglisi , 1999). 60A

steady state AP clamps( -contraction-) 25C 35C peak ICa ,

Ca influx . 60B (depleted) SR



146

(refilled) steay state (25C), AP ICa

. SR Ca ICa AP inactivation.

Ca- ICa inactivation .418 SR Ca (

peak ICa ), ICa 1 steady state( 10)

SR Ca release Ca influx (limit) .

60C 25C 35C ICa 12 ! 6 umol/L cytosol . , SR

Ca release ICa inactivation (net) Ca influx

~50% . , , square voltage clamp

pulse .419

SR Ca release ICa inactivation kinetics SR Ca release (timing)

(Puglisi , 1999; Linz Meyer, 1998). 60B(IDiff) 1

ICa trace (difference) L-type Ca SR Ca release-produced local [Ca]i index

.420 local [Ca]i (dIDiff/dt) SR Ca release locally sensed rate .

60D , Ca local SR Ca release peak 5 ms

, (release amplitude) (35C 2.5 ms). global

cellular Ca transient , RyRs Ca L-type Ca

. Ca-sensitive ionic

currents local [Ca]i sensor .

MODULATION OF ICa BY AGONISTS AND ANTAGONISTS

L-type Ca DHP421 (sensitivity) . ,

L-type Ca 1 DHPs

. DHPs ICa Ca channel blockers Ca channel antagonists

. DHPs, ()Bay K 8644, (+)S-202-791, CGP 28392 Ca channnel agonists

single Ca channel conductance opening duration

ICa .422 Bay K 8644 channel open time ~0.6 ms ! ~20 ms ,

Hess (1984a) Bay K 8644 Ca mode 2

opening ( 61). Bay K 8644

opening (normal state: mode 1) , mode 1 trace Bay K

418 ) This is an obvius consequence of more profound Ca-dependent inactivation of ICa. 419 ) Adachi-Akahani , 1996; Sham , 1995a; Trafford , 1997; Terraciano MacLeod, 1997; Linz

Meyer, 1998 420 ) We took the difference in ICa traces between pulse #1 and the other pulses in Fig 60B(IDiff) as an index of

the local [Ca]i produced by SR Ca release, as sensed by the L-type Ca channnel. 421 ) , nifedipine, nitrendipine, nimodipine, nisoldipine, isradipine PN200-110, Bay K 8644, azidopine,

iodipine . 422 ) Brown , 1984; Hess , 1984a; Kokubun Reuter, 1984; Kokubun , 1986



147

trace . Mode 2 type openig ,

(Hesss , 1984a; Yue , 1990). , Ca agonists mode 2 opening

. , Ca antagonist DHPs (mode 0)

ICa . Mode (switching)

time scale( ) .423 Ca

(mode 0), 0.15 ms

opening (mode 0a ; Yue , 1990), 0.5-5 ms bursts mode

(mode 1), mode 2(10-20 ms opening )

.424 Ca gaitng modal model ,

(Lacerda Brown, 1989).

Bay K 8644 long opening voltage clamp pulse ICa inactivation

. ICa , Ca influx

Ca- inactivation . Bay K 8644 -adrenergic

agonists( mode 2 enhance) ICa inactivation (dramatcally)

423 ) Switching between modes occurs on a slower time scale than bursts of activity(several second). 424 ) Indeed, normal Ca channels can be quiecent for many seocnds of depolarizing pulses(mode 0), can then

have very infrequent and brief opening of 0.15 ms(mode 0a), swtch to a mode where occasional bursts of 0.5-1 ms occur(mode 1) and rarely make excursions to mode 2(where openings of 10-20 ms are observed).



148

(Tsien , 1986; Taiho , 1990). Bay K 8644 ICa , tail

currents . Tail currents Ca ( negative Em deactivation

) Ca driving force . 61B Bay K

8644 sweep single channel tail Ca current . , Bay K 8644

activation/inactivation (10-20 mV) (negative) Em . Non-DHP Ca

channel antagonist FPL-64176 activation/inactivation Em Bay K 8644

tail ICa , DHP binding site (Rampre Lacerda,

1991; Kunze Rampre, 1992). Bay Y 5959 DHP Ca agonist(Bechem , 1997) tail

ICa ICa activation ( FPL-64176 ).

cardiac microsome425 nitrendipine ICa block

1000 (Bellemann , 1981; Lee Tsien, 1983). DHP-Ca channel

binding (voltage dependence) . Bean(1984)

Em 80 ! 10 mV nitrendipine ICa Kd 1000

, ~500 nM ! 0.36 nM ( 62). Sanguinetti Kass(1984)

, DHP (preferentially) .

sarcolemma vesicle 3H-DHP binding (Schilling Drewe, 1986) (Green ,

1985; Kokubun , 1986) . Hondeghem Katzung(1977)

Hille(1877) (local anesthetics) voltage-, use-dependent block of Na channels

modulated receptor hypothesis .

425 ) cardiac microsome heart tissue whole homogenate fraction . membrane microsome .



149

Sanguinetti Kass(1984) pH DHPs(nitrendipine, nisoldipine)

pH 7.4 (net charge) (verapamil, 426 nicardipine)

. (charged ligands) block (, voltage

pulse ) use-dependent. (receptor site)

. , (neutral ligands)

inactivated state ICa block (, holding potential

427), voltage-dependent use-dependent .

(hydrophobic nature) receptor

site . DHPs

DHPRs (Herbette , 1989; Valdivia Coronado,

1988). DHPs (,

) .428

. (benzodiazepine) Ca

blockers(; diltiazem) (Hering , 1993a,b).

(phenylalkylamines; Ca blocker , ; verapamil) SL

, (impermeant lignads)

426 ) to an intermediate degree 427 ) i.e. at depolarized holding potentials without requiring pulses 428 ) Kass Arena, 1989; Kass , 1991; Strubind , 1993



150

.429

DHPs voltage-dependence, use-dependent verapamil DHPs

(vasodilators) efficacy . , resting state

(vascular smooth muscle) Em ,

DHPs Ca Ca . Ca

(theraputic concentrations) DHPs block

. , Ca antagonists

pacemaker cells (profound) . Pacemaker

(diastolic) Em . Ca blockers

(antiarrhythmic effect) .

(class) L-type Ca : 1) DHPs

( ), 2) (AAs, verapamil, D600, D888, D890), 3) (BTZs

429 ) Hescheler , 1982; LeBlanc Hume, 1989



151

; diltiazem), allosterical ()( 63A,

Glossman , 1984, 1985). DHP binding

(stimulate), AA binding DHP binding binding (reciprocally) .

Ca 2 DHP binding , AA binding

(depress). Antagonists agonists DHPRs

(Kokubun , 1986; Brown , 1986), DHPs .

Ca agonists (stereoisomers) Ca antagonists (()-R-202-791 (+)

Bay K 8644) .430 , Ca agonist ((+)-

S-202-791) netagive test potential agonist Em = ~0 mV ICa antagonist

(, PN200-110 binding -allosteric enhancement-

-competitive inhibition- ; Kokubun , 1986; Kamp , 1989). Ca antagonist DHP

nitrendipine negative holding potential ICa (Brown

, 1986).431 , DHPRs , Ca

modulation . (diphenylbutylpiperidine)

neuroleptics (; fluspiriline pimozide) (benzoylpyrrole) Ca antagonist FPL-64176 Ca

.432 433

DHPs, AAs, Ca 1C

. 3H-azidopine reactive ligands photoaffinity labeling

, IIIS6, IVS6 DHP, BTZs .434 AA

IVS6 . 1C DHP (; 1A)

gain of function

(Grabner , 1996). Sinnegger (1997) 1C 9 non-conserved

a.a.(IIIS5, IIIS6, IVS6 ) 1A DHP (

51A, 63B). 1C alanine 1E/B loss of function

(approach) 1C DHP binding 13 (IIIS5 2, IIIS6 7, IVS6

6; Peterson , 1996, 1997; Schuster , 1996) (complementary data)

. AAs BTZs IIIS6 IVS6

(key sites) ( 63B, Hockman , 1997; Hering , 1996). , antagonist binding

435

(overlap). S5-S6 regions selectivity filter pore lining

430 ) Williams , 1985; Franckowiak , 1985; Kokubun , 1986 431 ) Even nitrndipine, a DHP known as Ca antagonist, can increase ICa activated from negative hilding

potentials. 432 ) ..may also bind to the Ca channel at an independent receptor. 433 ) Gould , 1983; Galizzi , 1986; Kunze Rampe, 1992 434 ) Streissnig , 1990a, 1991; Nakayama , 1991; Kraus , 1996 435 ) binding competition



152

activation gate regions ( 38 39).

-ADRENERGIC MODULATION OF CARDIAC Ca CURRENT

-adrenergic agonists ICa (classic observation)

(; Reuter, 1967). PKA , basal ICa 2-4

, /inactivation (negative) Em (shift)( 64A-B).436

Activation gating ICa negative Em ,

(negative) Em (maximal) ICa . Single channel PKA unitary conductance

, blank sweeps( opening ) open time

mode 2 gating (Cachelin, 1983; Yue , 1990).

-adrenergic cascade (sinaling pathways) ICa modulation

. , (ACh) basal ICa

(cathecholamine) (forskolin) ICa antagonize()(Fischmeister

Hartzell, 1986; Hescheler , 1986). ACh Gi protein muscarinic receptor

adenylyl cyclase (net) cAMP (Lindeman

Watanabe, 1989). , ACh cGMP , cGMP-activated

phosphodiesterase(PDE-II) cAMP (Fischmeister Hartzell, 1987),

cGMP-inhibited PDE III cAMP (Ono Trautwein, 1991; McDonald

, 1994). , ACh basal ICa 437 atrial latent pacemaker cells ACh

(withdrawal) ICa amplitude overshoot (Wang Lipsius, 1996).

cAMP , ICa (rebound)

(transient) post-vagal tachycardia . cGMP cGMP-

dependent protein kinase (PKG) , basal ICa

(ACh cGMP) cAMP ICa antagonize (Ono Trautwein,

1991). Nitric Oxide (NO) cGMP (Balligand , 1993)

ICa .438 NO cAMP-mediated ICa ACh-

mediated antagonize (Han , 1994a; Wang Lipsius, 1995b). ACh

protein phosphatase , PKA (mediated)

(reverse) (Herzig , 1995).

65 -adrenergic agonist pathway . Agonisit -adrenoreceptor439

436 ) Tsien , 1986; Hartzell, 1988; McDonald , 1994 437 ) basal cAMP . 438 ) Mery , 1993; Kristein , 1995; Han , 1994a 439 ) -adrenoreceptor -adrenergic, -adrenoreceptor -adrenergic -AR, -AR .



153

(occupation) GTP-binding protein(Gs) . Gs adenylyl cyclase

, cAMP . cAMP cAMP-dependent protein kinase(PKA) regulatory

subunit catalytic subunit , PKA catalytic subunit L-type Ca, RyRs, photpholamban, troponin

I (phosphorylation).

ICa -AR pathway() cAMP, cAMP analogs,

PDE inhibitors440 (forskolin) adenylyl cyclase 441,

(non-hydrolyzable) GTP analogs442 , PKA catalytic subunit

443 (mimick) . (protein

phosphatase 1, 2A) Ca (dephosphorylation) isoprenalinec ICa

(abolishes), basal ICa . () Ca

.

PKA Ca (vicinity) (anchored) ,

A kinase anchoring proein AKAP-79 (Gao , 1997a; Fraser , 1998).

Localization PKA- ICa

440 ) Tsien , 1972; Tsien , 1973; Vogel Sperelakis, 1981; Cachelin , 1983; Nargeot , 1983; Kameyama , 1985

441 ) Wahler Sperelakis, 1985; Hescheler , 1986 442 ) Josephson Sperelakis, 1978 443 ) Osterreider , 1982; Brum , 1983



154

(Zong , 1995). adenylyl cyclase cAMP , PKA

ICa ( 64), 1C

PKA inhibitor H-89 IBa (Perez-Reyes , 1994).

, 1C 2A PKA PKC (sunstrates)(Haase ,

1993; Puri , 1997). 1C (Ser-1928) AKAP- ICa ,

Ser-1928 C-terminal (distal part) Ca (fractoin)

(cleaved off)(Gao , 1997b). 2A nonconsensus PKA

(Ser-478, Ser-479) PKA ICa (Bnemann , 1999).

Gs ! ICa ,

(Yatani , 1987; Yatani Brown, 1989). , ( ) -AR

(Pelzer , 1990; MacDonald , 1994).

-agonist Ca channel gating (modify)? whole cell ICa (I) single

channnel current (i) : I = N po i , N

, po . Single channel conductance , i

(Reuter, 1982). Singel channel recording, -agonists dibutyryl-cAMP po

(Cachelin , 1983 Brum , 1984). N (

-dormant- ; Bean , 1984) -AR membrane patch

.444 N gating mode 0, mode 0a

mode1, mode 2 .445 , -AR ( ) ICa

Po . Po (Em-dependence)

mode channel gating .446

64A , PKA (negative) ICa ,

/inactivation Em-dependence . ICa

Em-dependence Ca channel gating current Em-dependence( -AR

; Bean, 1990) .447 Bean -stimulation Ca

ICa . , gating

Em (PO4)

surface potential effects ( 55). ,

.

444 ) There may also be some increase in N, but truly new channels do not appear in membrane patches with -stimulation(as would be expected if dormant channels become functional).

445 ) Yue , 1990; Cachelin , 1983; Brum , 1984 446 ) This increased Po is mediated either by a change in the Em dependence of activation or a shift toward

modes of channel gating where longer openings are favored. 447 ) This shift brings the Em-dependence of ICa activation closer to the Em-dependence of Ca channel gating

current(which is not shifted by -stimulation).



155

PKA ICa inactivation , ICa amplitude Ca-

inactivation . 64C-D ICa inactivation PKA-

, [Ca]o forskolin ( ICa

) PKA ICa inactivation . , Ca Ca-, Em-

inactivation , Ca influx SR Ca release

.448

1-, 2-AR agonists Gs (coupled) (inotropic

effects). , L-type Ca targets

.449 2-AR Gi-protein system (Zhou , 1999)450

. Mg ICa block , Mg-ATP

ICa (ORourke , 1992). () Mg-ATP

(maximal).

OTHER MODULATORS OF Ca CURRENT

(small) (confilicting),

Ca (modify) (McDonald , 1994). ,

-AR agonists ICa .451 , perforated patch recording

(phenylephrine) ICa

452(Zhang , 1998; Liu Kennedy, 1998). Whole cell voltage clamp 453

ICa rundown454 (limit) , ruptured-patch recording

(intracellular constituens) . Histamine H2

receptors , adenylyl cyclase cAMP cascade ICa

(Hescheler , 1987b; Levi Alloatti, 1988). Atrial natriuretic peptide (ANP) ICa

, cGMP cAMP .455 , ANP Gi

ICa cAMP cascade

448 ) Thus Ca channel phosphorylation slows both Ca-, and Em-dependent inactivation, but higher Ca influx and SR Ca release may functionally reverse this effect in the normal cellular environment.

449 ) Xiao Lakatta, 1993; Xiao , 1994b; Hool Harvey, 1997 450 ) 99 nature, sicence . .

451 ) Hescheler , 1988; Hartman , 1988; Ertl , 1991 452 ) .., phenylephrine induced a small transient decrease followed by a substantial increase in ICa only when

perforated patch recording. 453 ) perforate patch recording 454 ) sealing patch recording , ICa . current rundown , behavior feedback interaction in vitro .

455 ) Anand-Srivastava Cantin, 1986; Cramb , 1987; Gisbert Fischmeister, 1988; LeGrand , 1992



156

. , ANP guanylyl cyclase 456 cGMP PDE II cAMP

PKG ICa modulate . , cGMP basal ICa

(Ono Trautwein, 1991) PKG cardiac ICa

(contend)(Wahler , 1990; Sperelakis , 1996). Endothelin

ICa (Inoue , 1990), patch pipette GTP ICa

GTP ICa (enhanced)(Tohse , 1990; Lauer , 1992).

Perforated patch endothelin basal ICa , -

agonist ICa ETA pertussis-toxin senstive Gi

(reversed)(Thomas , 1997).

(adenosine) P1-purinergic receptor457 , Gi -agonists

ICa . , ACh basal ICa (Belardinelli

Isenberg, 1993b; West , 1986). P2- Gs ICa

(Scamps , 1992), (inhibitory effects) (; Qu , 1993).

Angiotensin II( II) ICa , PKA protein kinase C(PKC)

.458 Phorbol esters PKC

ICa 459, 460, (biphasic)461, ICa

.462 , Zhang (1997a)

phorbol ester PMA C2- PKC(PKC-, -, -) .

Protein tyrosine kianse(PTK) inhibitors (PTK , ; genestein) ruptured-patch ICa

.463 , PTK (larger) (slower)

perforated patch (Wang Lipsius, 1998).464 cytosolic PTK

, membrane associated465 PTK basal ICa .

Genestein PTK block ICa IK -agonist (Hool , 1998). ,

tyrosine kinases adrenergic signaling , kinase cascades crosstalk

.466 (arachidonic acid) (epoxyeicosatrienoic ecids; EET)

456 ) ANP receptor type A, B, C A, B membrane-bound guanylyl cyclase . 457 ) P1- ; P1-redeptors. cf. P2-receptors 458 ) Allen , 1988; Dsemeci , 1988; LeGrand , 1991 459 ) Dsemeci , 1988; Lacerda , 1988 460 ) Tseng Boyden, 1991; Scamps , 1992; Zhang , 1997a 461 ) Lacerda , 1988 462 ) Walsh Kass, 1988 463 ) Katsube , 1998; Hool , 1998; Ogura , 1999 464 ) , but also have a coexistent larger and slower stimulatory effect that is apparent in perforated patch

experiements. 465 ) cytosolic vs. membrane associated (membrane bound) : platform membrane targeting protein membrane associated .

466 ).. and reflects a crosstalk between these cascades.



157

cardiac ICa (Petit-JaquesHartzell, 1996; Chen , 1999a).

(phosphatase) , EETs inactivation single

channel Po conductance .

ICa regulation 467

. , Gs Gi( G proteins) cyclic nucloetides

(cAMP, cGMP) modulation , protein kinase (PKA, PKC, PKT, PKG) .

modulation

.468

, L-type ICa Ca (leak, Na/Ca exchange

ICa.T ), ICa cardiac E-C coupling Ca regulation,

. Action potential ICa kinetics amplitude SR Ca

(controlling) (critical factors)(8). ICa Ca SR Ca

stores (replenish) myofilaments (9 ).

ICa Ca cardiac cycle (NCX )

steady state . Ca influx (uncompensated)

Ca load . conductance , Ca

inactivation Ca gain469 (; Em window ICa

). (relaxation) (compromise)

(arrhythmogenic) .

467 ) conflict 468 ) Clearly additional work will be required to clarify the details of these intermingling pathways for many of

these important modulatory mechanisms. 469 ) . electrophysiology . Gain, loss, conductance .

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