Calcium Channels are Ganging up in the Sarcolemma

Navedo et al.¹ in this issue provide provocative and exciting data, indicating that up to ~6 L-type Ca²⁺ channels (LTCC, in this case Ca_v1.2) can physically interact with each other to gate in a coupled manner, producing large and long-lasting Ca²⁺ influx into smooth and cardiac muscle myocytes. This coupling appears to be relatively rare and transient, with a minority of overall LTCCs involved at any moment. However, coupling is favored by either displacement of calmodulin (CaM) from its binding location in the C-terminal LTCC domain, activation of PKCα, chronic hypertension (in vascular smooth muscle) and LTCC mutants that are associated with cardiac arrhythmias and hearing loss in humans (Timothy's Syndrome or LQT8).

The fundamental observations here were made using TIRF (total internal reflection fluorescence) microscopy by measuring local submembrane Ca²⁺ events termed Ca²⁺ sparklets.^2-4 These are attributable to Ca²⁺ entry via LTCC, unlike the SR/ER Ca²⁺ release events known as Ca²⁺ sparks or puffs (mediated by ryanodine or InsP₃ receptor channels). Physiologically Ca²⁺ sparklets would be extremely small and brief events, because normal LTCC openings are < 1 ms in duration and current amplitude is only ~0.2 pA (at physiological [Ca²⁺]_o) resulting in an integrated Ca²⁺ influx of only 0.1 fC (or 300 Ca²⁺ ions).⁵ This would produce a very small hard to detect fluorescence signal. However, the Ca²⁺ sparklets here are much larger, which enhances detectability. They are larger because they are due to more rare persistent openings (like mode 2 openings favored by the LTCC agonist Bay K 8644),⁶ elevation of [Ca²⁺]_o and measurements at negative voltages (where driving force is high). This group has previously shown using patch clamp that these Ca²⁺ sparklets are indeed caused by LTCC current (not SR Ca²⁺ release), can last hundreds of milliseconds and are favored by PKC activation.³ TIRF imaging has the advantage (for these events) that one can sample the entire surface of the cell simultaneously, rather than just a small patch of membrane in cell-attached patch clamp recording.

Here, they show that local Ca²⁺ sparklets and patch clamp currents show quantal increments of 2-6 times the normal Δ[Ca²⁺]_i and single Ca²⁺ current amplitude, and that the apparent gating transitions of each channel occur essentially simultaneously (i.e. coupled gating). This raises 3 obvious questions: 1) are these coupled gating events real, 2) why might they be of functional importance or relevance, and 3) how are they regulated?

Coupled gating of LTCC seems real

I find their cumulative evidence compelling,¹ especially because of the patch-clamp data, which has a similar phenotype as the Ca²⁺ sparklets and where channel opening and closing events are more discretely identifiable. It is also hard to imagine an artifact or technical limitation that would produce the striking coupled gating traces in Figure 2. The only thing missing is perhaps the absolute proof that these large currents (6 times the single LTCC current) are indeed due to LTCC and not due to some other higher conductance myocyte channel that contaminates the record. However, as these events are rare it may be harder to demonstrate clear nifedipine-sensitivity (as they have done for normal Ca²⁺ channel openings). On this point (and in light of the other data), I am inclined to give them the benefit of the doubt. The conclusion that the large events are indeed coupled seems inescapable, because the relatively rare single channel events would virtually never superimpose, and as the authors point out, coupled events very far outweigh that coincidental possibility. Moreover, Ca²⁺ entry through one LTCC would be expected to inhibit (rather than activate) a nearby LTCC (via Ca²⁺-dependent inactivation),⁷ making this sort of chemical coupling very unlikely. Note that this is in striking contrast to coupled gating reported for SR Ca²⁺ release channels, RyR)^8,9 where controversy still remains, in part because it is known that Ca²⁺ release from one RyR will tend to activate its neighbors extremely rapidly, via Ca²⁺-induced Ca²⁺-release.¹⁰ Note that LTCCs in smooth and cardiac muscle do not exhibit the paracrystalline array and neighbor abutment seen for RyR in striated muscle or Ca_v1.1 channels in skeletal muscle.¹¹ Finally, the FRET studies here suggest (but do not prove) direct physical interaction between LTCC monomers. They also led the authors to an intriguing initial working hypothesis that LTCCs interact via a C-tail to C-tail interaction. It will be interesting to see if this model holds up upon further study. The LTCC C-terminal is a very busy regulatory zone, and this is an exciting new twist.

Why might coupled LTCC gating be of functional importance or relevance?

The effect of coupled gating would be to increase the synchronous local Ca²⁺ influx. While not addressed yet, this physical coupling could also increase the duration of opening, because one coupled channel may not as readily close if the others are still open. This might be an interesting angle for some follow-up studies. In any event, a larger local Ca²⁺ influx might be advantageous in assuring local signaling fidelity. For example where LTCC influx triggers SR Ca²⁺ release via RyR, a larger and longer Ca²⁺ influx would ensure SR Ca²⁺ release from each site. On the other hand, it has been argued that even a single LTCC opening, which would raise cleft [Ca²⁺]_i to ~10 μM (in tens of μs), may suffice to ensure the fidelity of E-C coupling in the heart, especially where multiple LTCC at the site each have a chance to independently activate the local RyR.^5,12-14 And with 20,000 SR junctions needing to fire at every heartbeat, the energetic cost of extruding extra Ca²⁺ that enters might favor keeping the local LTCC entry modestly above that required for high fidelity. My suspicion is that this coupled LTCC gating with large locally synchronized Ca²⁺ entry may be most important for specific loci involved in the activation of local Ca²⁺ targets that require either very high or very sustained local [Ca²⁺]_i to assure signaling fidelity. Identifying these specific Ca²⁺ targets and roles that require this sort of focal high [Ca²⁺]_i signal would be a valuable extension of this work.

Moreover, if this apparently rare situation is to be physiologically important (as opposed to a biophysical curiosity) it must be influential to at least one Ca²⁺ signaling mechanism. This does not mean that a majority of the cell's LTCC would have to be recruitable to these clusters. Indeed, it may turn out that this clustering and coupling is driven by regulation discussed below to a locus where a small minority of LTCC in the cell join for high fidelity discrete local Ca²⁺ signaling (independent of whole cell Ca²⁺ signals that activated muscle contraction).

How is coupled gating of LTCC regulated?

On this point the authors already have several very intriguing leads, and they have tried to tie this in to the structure of the LTCC as well. CaM (that mediates Ca²⁺-dependent inactivation) is thought to be normally bound to the LTCC,¹⁵ and that limits coupled gating.¹ However, if the LTCC loses CaM, coupled gating is dramatically augmented. So there may be some finite fraction of LTCC that lack CaM and this may be regulated somehow, especially since there is intense competition among cellular sites for a limited pool of CaM (even in the unbound state).¹⁶ They also have data to suggest that the mechanism by which PKCα activates coupled gating is that it dislodges CaM from the LTCC (although that cannot be an obligate mechanism, because the CaM inhibitor W7 still enhances coupled gating when PKCα is knocked out). The anchoring protein AKAP150 also seems to enhance coupled gating, perhaps by bringing key signaling molecules to a subset of LTCCs at specific foci.

Finally, they show that coupled gating seems to be more prominent in two pathophysiological models. Vascular smooth muscle cells from angiotensin-II induced hypertensive rats (which may tie into the PKCα effect above) show enhanced coupled gating, which could conceivably contribute to the clinical phenotype. The Timothy Syndrome mutant LTCC also exhibited more coupled gating, weaker CaM association with the channel, and more C-tail to C-tail FRET than WT channels. It remains to be seen if the coupled gating and homo-multimerization of LTCC α subunits really contributes to these clinical phenotypes, but the direction of the effects is right. These new results are provocative, and further studies will be required to further validate and understand the potential physiological and pathophysiological importance of this phenomenon. However, Navedo et al.¹ have gotten this idea off to an exciting start here.

Non-standard Abbreviations and Acronyms

LTCC

L-type Ca channel

PKC

protein kinase C

LQT

Long QT syndrome

SR

sarcoplasmic reticulum

ER

endoplasmic reticulum

CaM

calmodulin

TIRF

total internal reflection fluorescence

InsP₃

Inositol 1,4,5 trisphosphate

RyR

ryanodine receptor

FRET

fluorescence resonance energy transfer