Dissecting the functional role of different isoforms of the L-type Ca²⁺ channel

There currently exist a great number of different mouse lines in which the activity of a particular gene of interest has been inactivated or enhanced. However, it is also possible to insert specific mutations in a gene so that the pharmacological sensitivity of the gene product is altered. An example of such an approach shows how the abolition of the sensitivity of an L-type Ca²⁺ channel isoform to dihydropyridines allows the investigation of the physiological role of these channels in different tissues .

L-type Ca²⁺ channels (LTCCs) are formed by different pore-forming α1 subunit isoforms named Ca_v1.1, Ca_v1.2, Ca_v1.3, and Ca_v1.4 associated to auxiliary subunits (α2-δ, β, and γ) (1). The common pharmacological hallmark of all native and recombinant LTCCs is their sensitivity to dihydropyridines (DHPs). However, the small differences among the LTCC α1 isoforms in their affinity for DHPs (agonists and antagonists) have limited the study of the functional role of these channels in various tissues, including the cardiovascular system, the brain, and the endocrine glands.

In this issue of the JCI, Sinnegger-Brauns and coworkers report that they have developed a new mouse model resulting from a knock-in mutation of the Ca_V1.2 voltage-dependent LTCC subunit which abolishes the sensitivity of the channel to DHP (referred to herein as the Ca_V1.2DHP^–/– mouse) (see Figure 1) (2). Since Ca_v1.2 is almost ubiquitously expressed in excitable cells, the model provides a very elegant and promising approach for investigating the physiological role of the other DHP-sensitive LTCC isoforms (Ca_v1.1, Ca_v1.3, and Ca_v1.4). This model is particularly useful for the exploration of the function of Ca_v1.3 channels, which are coexpressed with Ca_v1.2 but at a much lower level in most cell types investigated so far. The Ca_v1.1 subunit is responsible for excitation-contraction coupling in skeletal muscle (3), while Ca_v1.4 expression appears to be restricted to the retina and T lymphocytes (4–6). In contrast to the tissue-specific expression of Ca_v1.1 and Ca_v1.4, both Ca_v1.2 and Ca_v1.3 are widely expressed and distributed in the brain, the cardiovascular system, and neuroendocrine cells.

Figure 1

LTCCs are multimeric complexes of subunits formed by an α1 pore-forming protein associated to three auxiliary subunits (α2-δ, β, and γ). Four genes encode the pore-forming subunit (Ca_v1.1_Ca_v1.4). The primary structure, α1, is represented in the figure showing the four domains composed of six transmembrane segments. Schematic representation of the knock-in mutation in the Ca_V1.2 gene (Thr1066 to Tyr) realized by Sinnegger-Brauns and coworkers in this issue of the JCI (2) to discriminate in vivo the contributions of Ca_V1.2 and Ca_V1.3 to various physiological functions.

Using the Ca_V1.2DHP^–/– mice that exhibit a normal phenotype in spite of their DHP-insensitive Ca_v1.2 channels, the authors have been able to differentiate, both in vivo and in vitro, the roles of the Ca_v1.2 and Ca_v1.3 channels in insulin secretion, cardiac performance, and mood behavior (2). In particular, the finding that insulin secretion in these mice was completely insensitive to DHP agonists and antagonists provides strong evidence in the long-standing debate about the insulin secretagogue role of the Ca_v1.2 and Ca_v1.3 channels. Opposite conclusions about the importance of the Ca_v1.3 LTCC in this secretion process have been drawn by two previous studies using different mouse strains lacking the Ca_v1.3 LTCC (7, 8) and a β-cell–specific knockout of the Ca_v1.2 LTCC (9). The complete resistance of the insulin secretory process to DHPs in the Ca_V1.2DHP^–/– mice strongly supports the minor role of Ca_v1.3 in this process.

Another important aspect of the study concerns the role of the Ca_v1.3 LTCC in integrated neuronal functions. While the expression of the Ca_v1.2 LTCC isoform accounts for about 80% of the overall DHP-binding sites in the brain, the remaining 20% of binding can be attributed to Ca_v1.3 LTCCs, and little was known about the function of these channels. Indeed, DHP agonists such as BayK8644 cannot be used in vivo because of potent neurotoxic effects mediated by Ca_v1.2 channels. Abolition of the sensitivity of the Ca_v1.2 channel to DHPs allowed unmasking of a depression-like behavioral effect mediated by Ca_v1.3 channels (2). From a molecular point of view, these effects may rely on the recently identified specific interactions of the Ca_V1.3 channel with synaptic proteins (10), with possible consequences related to the control of neurotransmitter release classically implicated in depression (as shown by Sinnegger-Brauns et al. in the case of dopamine). Therefore, an attractive prospective application of these findings is in the development of new classes of antidepressant drugs that act selectively on Ca_v1.3 channels.

The Ca_V1.2DHP^–/– mouse model can also help to reveal the functional role of LTCCs in the physiology of the heart. It is widely accepted that the Ca_v1.2 subunit constitutes the most abundant LTCC subunit in the heart, where it plays an important role in excitation-contraction coupling in the working myocardium. In addition, a growing body of evidence indicates that the functional roles of Ca_v1.2 and Ca_v1.3 channels are distinct in the heart, with Ca_v1.3 channels playing a major role in pacemaker activity. Indeed, the fact that mice in which the gene encoding the Ca_v1.3 subunit has been inactivated show prominent dysfunctions in pacemaker activity in vivo and in vitro demonstrates that Ca_v1.3 channels constitute the major component of the L-type current in pacemaker cells (8, 11, 12). Experimental results presented by Sinnegger-Brauns and coworkers clearly support this view (2). More generally, the use of engineered mice such as Ca_V1.2DHP^–/– mice will be of particular interest to further assess the contribution of the different ionic currents underlying diastolic depolarization in sinoatrial pacemaker cells. Automaticity in cardiac pacemaker cells is due to the slow diastolic depolarization phase, which drives the membrane voltage from the end of the action potential to the threshold of the following action potential. Both native and recombinant Ca_v1.3 channels exhibit more negative threshold for activation and slower inactivation kinetics than Ca_v1.2 channels. In other words, the low threshold of Ca_v1.3 current is consistent with a major role during diastolic depolarization (12), while a sustained calcium influx is required for the contractility of heart cells.

In conclusion, the originality of this model offers stimulating prospects for dissecting the physiological roles of calcium channels in various tissues (2). This genetic “reverse” pharmacology in vivo is likely to be applied in the future to other channels and receptor families sharing a similar pharmacology.

See the related article beginning on page 1430.

Nonstandard abbreviations used: dihydropyridine (DHP); L-type Ca²⁺ channel (LTCC).

Conflict of interest: The authors have declared that no conflict of interest exists.

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