Hyperpolarization- and cyclic nucleotide-activated (HCN) channels contribute to rhythmic oscillations in excitable cells. They possess an intrinsic autoinhibition with a hyperpolarized V 1/2, which can be relieved by cAMP binding to the cyclic nucleotide binding (CNB) fold in the C-terminal region or by deletion of the CNB fold. We questioned whether V 1/2 shifts caused by altering the autoinhibitory CNB fold would be accompanied by parallel changes in activation rates. We used two-electrode voltage clamp on Xenopus oocytes to compare wildtype (WT) HCN2, a constitutively autoinhibited point mutant incapable of cAMP binding (HCN2 R591E), and derivatives with various C-terminal truncations. Activation V 1/2 and deactivation t 1/2 measurements confirmed that a truncated channel lacking the helix αC of the CNB fold (ΔαC) had autoinhibition comparable to HCN2 R591E; however, ΔαC activated approximately two-fold slower than HCN2 R591E over a 60-mV range of hyperpolarizations. A channel with a more drastic truncation deleting the entire CNB fold (ΔCNB) had similar V 1/2 values to HCN2 WT with endogenous cAMP bound, confirming autoinhibition relief, yet it surprisingly activated slower than the autoinhibited HCN2 R591E. Whereas CNB fold truncation slowed down voltage-dependent reaction steps, the voltage-independent closed-open equilibrium subject to autoinhibition in HCN2 was not rate-limiting. Chemically inhibiting formation of the endogenous lipid PIP2 hyperpolarized the V 1/2 of HCN2 WT but did not slow down activation to match ΔCNB rates. Our findings suggest a “quickening conformation” mechanism, requiring a full-length CNB that ensures fast rates for voltage-dependent steps during activation regardless of potentiation by cAMP or PIP2.
The Journal of Membrane Biology – Springer Journals
Published: Jun 30, 2015
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera