In this work, Morgenstern and colleagues describe an approach involving functionalized nanobodies which decrease the activity of voltage-gated Ca2+ channels associated with β1 subunits and promote their removal from the surface membrane of neurons and muscle.
Voltage-gated Ca2+ channels are composed of multiple subunits and are broadly expressed in excitable cells
Voltage-gated Ca2+ (CaV) channels are broadly expressed in excitable cells throughout the body, where they regulate multiple physiological processes, including cardiac, skeletal, and smooth muscle contraction as well as neuronal excitability, neurotransmitter release, and gene expression. Accordingly, CaV channels are a major target for the treatment of cardiovascular and neurological disease. Morgenstern and colleagues1 describe an approach for controlling the function of CaV channels with a specific molecular composition.
CaV channels are formed by one of ten pore-forming α1 subunits, which largely determine their conductive properties. Four of these subunits contribute to L-type Ca2+ channels (CaV1.1-4), while R-, N-, and P/Q-type channels (CaV2.1-4) are composed of their own distinct α1 subunits.
The CaV1 and CaV2 subfamilies associate with a range of auxiliary subunits: β, encoded by CACNB1-4; α2δ, encoded by CACNA2D1-4; and γ, encoded by CACNG1-82,3. α1 subunits form a strong interface with β subunits at the intracellular α-interaction domain of the channel4,5. It is believed that CaV1 and CaV2 channels all assemble with a single β subunit, which serves to promote membrane expression and set channel voltage-dependencies. Expression of β1-4 subunits has been detected in the brain. β1 is also expressed in skeletal muscle, whilst β2 subunits are expressed in heart, lung, and smooth muscle. Finally, β3 subunits are expressed highly in smooth muscle.
The β subunit prevents ubiquitination of the α1/β channel complex, increasing membrane channel density and whole-cell current magnitude6. β subunits also confer diverse biophysical properties to the mature CaV channels, enabling G protein regulation, setting rates of channel activation and inactivation, and fine-tuning voltage dependency7. β subunits also serve as sites for post-translational modification and protein-protein interactions. For example, palmitoylation allows β2a to embed into the plasma membrane, slowing the rate of channel inactivation8. More recently, adrenergic stimulation of CaV1.2 in the heart was shown to involve an interaction between β2a and the Rad G protein, which becomes phosphorylated by protein kinase A to relieve its constitutive inhibition of CaV1.29.
Most CaV channel agonists and antagonists work by binding to their α1 subunits. However, because of the broad expression of CaV channels, this pharmacological approach does not afford precise, tissue-specific regulation of Ca2+ entry. Gene ablation or siRNA-mediated protein knockdown of β or other subunits approaches could circumvent these limitations, but the interpretation of these experiments is confounded when there are multiple auxiliary subunit isoforms expressed in a cell, some of which have partially overlapping functions.
Nanobodies target ion channels of specific composition with precision
Here, Morgenstern et al.1 describe an elegant and highly effective strategy to decrease the functional impact of β1-associated CaV channels using nanobodies.
Nanobodies are recombinant antigen binding fragments, and their small size and folding properties enhance their stability inside of live cells, where they can be utilized as “intrabodies”10. Morgenstern et al.1 demonstrate how CaV channels may be targeted with functionalized nanobodies, to precisely inhibit channels comprising of specific β subunit isoforms.
In their previous work, Morgenstern et al.11 demonstrated a CaVβ-targeted nanobody (nb.F3) inhibits CaV1/2 channels by initiating their redistribution into endosomes. This nanobody-delivered ubiquitination machinery (CaV-aβlator) functions as an effective inhibitor of CaV channels. In this present work, Morgenstern et al.1 reveal a refined inhibitor specifically targeted to β1-associated CaV channels (Chisel-1).
Briefly, the authors identified a nanobody (nb.E8) which selectively binds the CaVβ1 SH3 domain and inhibits CaVβ1-associated voltage-gated CaV channels by decreasing open probability and increasing their rate of channel inactivation. Interestingly, nb.E8 also decreases channel activity by reducing channel surface density.
Functionalizing nb.E8 with the Nedd4L HECT domain yielded Chisel-1, which eliminated current through CaVβ1-reconstituted CaV1/CaV2 and native CaV1.1 channels in skeletal muscle. Chisel-1 also decreased depolarization-induced Ca2+ entry and excitation-transcription coupling in hippocampal neurons. Notably, Chisel-1 was ineffective against CaVβ2-associated CaV1.2 channels in cardiomyocytes, underscoring its specificity. In a therapeutic setting, genetically-encoded inhibitors like CaV-aβlator and Chisel-1 could be selectively expressed within cells of interest, potentially bypassing the off-target effects produced by many traditional CaV inhibitors.
Nanobodies could reveal important aspects of ion channel organization and function
The findings by Morgenstern et al.1 raise multiple intriguing questions. For example, how does binding of the nb.E8 nanobody to CaVβ1, independent of ubiquitin ligase conjugation, act to reduce the membrane surface density of CaV2.2 channels? Does the reduction in channel activity in the presence of nb.E8 prime the channel for endocytosis? Also, CaV channels form clusters in the surface membrane of neurons and muscle12,13. Recent studies indicate that ion channels involved in cooperative signaling cascades co-cluster. This raises the question of whether Chisel-1-bound channels are removed individually within a cluster or if the binding of a subset of channels destines the entire cluster for removal? The latter mechanism would suggest an amplification mechanism which could impact on clustered channels. Future studies should investigate these questions.
Morgenstern T. J. et al. Selective posttranslational inhibition of CaVβ1-associated voltage-dependent calcium channels with a functionalized nanobody. Nat. Commun. https://doi.org/10.1038/s41467-022-35025-7 (2022).
Dong, Y. et al. Closed-state inactivation and pore-blocker modulation mechanisms of human CaV2.2. Cell Rep. 37, 109931 (2021).
Wu, J. et al. Structure of the voltage-gated calcium channel Cav1.1 complex. Science 350, aad2395 (2015).
Davies, A. et al. The alpha2delta subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function. Proc. Natl Acad. Sci. USA 107, 1654–1659 (2010).
Pragnell, M. et al. Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit. Nature 368, 67–70 (1994).
Altier, C. et al. The Cavbeta subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels. Nat. Neurosci. 14, 173–180 (2011).
Canti, C., Bogdanov, Y. & Dolphin, A. C. Interaction between G proteins and accessory subunits in the regulation of 1B calcium channels in Xenopus oocytes. J. Physiol. 527, 419–432 (2000).
Chien, A. J., Carr, K. M., Shirokov, R. E., Rios, E. & Hosey, M. M. Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function. J. Biol. Chem. 271, 26465–26468 (1996).
Liu, G. et al. Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature 577, 695–700 (2020).
Dong J. X. et al. A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons. Elife 8, (2019).
Morgenstern T. J., Park J., Fan Q. R., Colecraft H. M. A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVbeta subunits. Elife 8, (2019).
Dixon, R. E., Navedo, M. F., Binder, M. D. & Santana, L. F. Mechanisms and physiological implications of cooperative gating of clustered ion channels. Physiol. Rev. 102, 1159–1210 (2022).
Sato, D. et al. A stochastic model of ion channel cluster formation in the plasma membrane. J. Gen. Physiol. 151, 1116–1134 (2019).
This work was supported by grants from the NIH (HL152681, NS114210, and 1OT2OD026580).
The authors declare no competing interests.
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Manning, D., Santana, L.F. Regulating voltage-gated ion channels with nanobodies. Nat Commun 13, 7557 (2022). https://doi.org/10.1038/s41467-022-35027-5