Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations.
OBJECTIVE
The hyperpolarization-activated cyclic nucleotide-gated (HCN) current I(f)/I(HCN) is generally thought to be carried by Na(+) and K(+) under physiological conditions. Recently, Ca(2+) influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca(2+) permeation through I(f)/I(HCN) is still lacking.


RESULTS
To possibly provide direct evidence of Ca(2+) influx through I(HCN)/I(f), we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human I(f), since Ca(2+)-mediated I(f)/I(HCN) currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca(2+) inward currents upon hyperpolarization with a single-channel amplitude of -0.87+/-0.06 pA, a low open probability of 3.02+/-0.48% (at -110 mV, n=6, Ca(2+) 2 mmol/L), and a Ca(2+) conductance of 8.9+/-1.2 pS. I(HCN2-Ca2+) was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific I(f) inhibitor ivabradine, clearly confirming that Ca(2+) influx indeed was conducted by HCN2 channels. Changing [Na(+)] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca(2+) caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na(+)/Ca(2+) conductance, whereas the K(+) conductance of HCN2 increased significantly in the absence of external Ca(2+) with increasing K(+) concentrations. The mixed K(+)-Ca(2+) conductance, however, was unaffected by the external K(+) concentration. Notably, we could also record hyperpolarization-activated Ca(2+) permeation of single native I(f) channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca(2+) conductance of human I(f), 9.19+/-0.34 pS; amplitude, -0.81+/-0.01 pA; open probability, 1.05+/-0.61% at -90 mV).


CONCLUSIONS
We directly show Ca(2+) permeability of native rat and, more importantly, human I(f) at physiological extracellular Ca(2+) concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased I(f) density and HCN expression.
Michels
Guido
G
Department of Internal Medicine III, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany.
Brandt
Mathias C
MC
Zagidullin
Naufal
N
Khan
Ismail F
IF
Larbig
Robert
R
van Aaken
Sebastian
S
Wippermann
Jens
J
Hoppe
Uta C
UC
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000831:Animals, Newborn; D001552:Benzazepines / Q000494:pharmacology; D016466:CHO Cells; D020013:Calcium Signaling; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D000077550:Ivabradine; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D001552:Benzazepines; D054815:Cyclic Nucleotide-Gated Cation Channels; C578710:HCN2 protein, human; C578711:Hcn2 protein, mouse; C578712:Hcn2 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D000077550:Ivabradine; D012964:Sodium; D000242:Cyclic AMP; D011188:Potassium