Voltage-Dependent K⁺ (K_V) Channels: A Large and Diverse Family of Membrane Voltage Regulators

Introduction

K⁺ selective channels are some of the most widespread ion trafficking molecules in living organisms, with more than 70 genes encoding different K⁺ channels in humans.

K⁺ channels are gated by a variety of factors: voltage, cyclic nucleotides, ATP, Ca²⁺, Na⁺ and G-proteins, which may act singly or in combination. Therefore, many of these channels serve and operate as sensors for the cell’s metabolic state. In addition, K⁺ channels contribute to maintenance of a cell’s resting membrane potential and fine-tuning excitability in neurons, heart and muscle, mainly by defining the action potential duration and the intervals between them.

Naming K⁺ Channels

Traditionally, K⁺ currents were described and divided according to their function mainly in excitable cells. These include the classical delayed rectifier (DR), transient A-type, inward rectifier (IR), Ca²⁺ dependent and leak currents¹.

The current classification of K⁺ channels relies on molecular biology and gene homology. The primary classification divides K⁺ channels into voltage-dependent K⁺ (K_V) channels (a super family that includes also the EAG and KCNQ families), Ca²⁺-dependent K⁺ (K_Ca) channels, inward rectifier (K_ir) and two-pore domain K⁺channels² (see Figures 1 and 2 for schematic structures).

Here we describe the classification, structure and some roles of the K_V super family of channels. 38 genes encoding members of the K_V channel super family have been described to date in the human genome^3,4. Most can be grouped into six families while some others may be defined as electrically silent K_V α subunits (see Table 1). K_V channels fall into one of the two classical categories of DR and A-type.

Delayed rectifier was the original name attributed to voltage-dependent K⁺ channels due to their delayed activation in squid giant axons (relative to the fast-activating Na⁺ channels). Members from all K_V subfamilies (including K_V1-4, EAG and KCNQ) can form delayed rectifier channels¹.

A-type channels are low voltage-activated, fast-inactivating (therefore, transient) K⁺ channels. A-type channels are usually composed of members of the K_V1 and K_V4 subfamilies and the auxiliary β subunit is often necessary for the fast-inactivation phenomenon^5,6.

Recently, a novel auxiliary subunit (CD26- related Dipeptidyl Aminopeptidase-like Protein, DPPX) was shown to confer neuronal A-type characteristics to K_V4 channels⁷.

Structure of K_V Channels

K_V channels are composed of two types of protein subunits: the α subunit, which forms the pore and auxiliary (β) subunits. α subunits tetramerize in order to form a functional K⁺ conducting channel⁸.

The K_V α subunit is a protein with six transmembrane (TM) α helixes (TM1-6 or S1-6) and intracellular (cytoplasmatic) N- and C- termini. The most defined structural features are the extracellular loop between the fifth and sixth TM (which forms the K⁺ selectivity filter and the channel pore) and the fourth TM, which is positively charged and forms the voltage sensor of the protein (see Figure 2).

In addition, many functions were attributed to the usually large N- and C- termini loops. These include control of the activity dependent regulation of channel closure (inactivation), protein-protein interactions (such as tetramization of α subunits, interaction with the cytoplasmic β subunits and ER retention sequences⁹)⁸. K_V α subunits can form either homotetramers or heterotetramers, probably restricted to the same subfamily (as in Table 1). The huge diversity of K_V currents in vivo is due to: existing splice variants (of the same α subunit isoform gene), assembly of the α subunits tetramer with different auxiliary subunits and heteromerization of α subunits.

Regulators of Excitability

The K⁺ ion is not equally distributed between the cytoplasm of cells and the extracellular space, leading to a strong driving force for K⁺ efflux over most of the physiological range of membrane voltages¹. K_V channels open in response to depolarization of the transmembrane voltage and facilitate such effluxes. Depolarization can be described as accumulation of positive charges on the outer phase of the membrane. However, efflux of a positively charged ion (such as K⁺) will drive back the membrane potential toward the resting (polarized) potential. Thus, K_V channels are molecules that sense depolarization and, in turn, act to cancel it.

One of the most well characterized roles of K_V channels is to terminate the strong depolarization caused by activation of voltage-dependent cation influx (together forming the action potential waveform). Therefore, one can postulate that any increase in K_V channel activity will lead to more efficient termination of depolarization (usually shortening or eliminating action potentials) and vice versa. This functional ability of K_V channels also leads to determination of the action potential firing frequency^10,11. Thus, K_V channel activity plays a key role in neuronal coding.

The mere existence of 38 genes, many more gene products and even more types of functional K_V channels, demonstrates that there are many ways for cells to terminate depolarization once it occurs.

Other Roles of K_V Channels

K_V channels interact with other proteins and molecules to modulate the channels function. In this way K_V channels “sense” these agents and respond in a typical way. For example, the K_V2.1 channel (probably when in a heteromeric form together with the “silent” K_V9.3 subunit) contributes to an O₂-sensitive K⁺ current in pulmonary arteries, which is responsible for vasoconstriction following hypoxia¹². In the HEK cells expression system, K_Vβ1.2 confers O₂-sensitivity to K_V4.2 channels, which suggests a role in response to hypoxia¹³.

Toxins and Antibodies as Molecular Tools to Study K_V Channels

Due to the huge variety in the types of functional K_V channels in vitro, it became very important to identify the molecular entities corresponding to native K_V currents in different tissues, cell types and developmental stages. Therefore, molecules that can bind K_V channel components specifically were demonstrated to be powerful tools in characterizing both the channel structure and its physiological role. Two main groups of such molecules are peptidyl toxins and specific antibodies.

Peptidyl toxins (isolated from animal venoms) represent a group that modulate channel function (see an example in Figure 3) and therefore alter native responses. In addition, peptidyl toxins facilitate specific detection^14,15. Specific K_V toxins are often used to dissect the particular contribution of different subunits to native currents^10,11,16 (or some other physiological phenomena derived from or dependent on K_V currents¹⁷) and in determining the heteromeric composition of K_V channels^10,16,18. Recently, it was shown that fluorescent-labeled specific toxins were used for localization of K_V1.3 channels in T lymphocytes¹⁹.

Antibodies designed to bind specific K_V channel isoforms are mainly used in localization and colocalization studies^16,20-28. However, specific antibodies were demonstrated to act as functional blockers of K_V1.2, K_V1.5 and K_V2.1 (our catalog numbers: #APC-010, #APC-004 and #APC-012 respectively) in smooth muscle cells. In this work, the authors took advantage of the cytoplasmic epitope of the designed antibodies and the use of the whole cell patch clamp method to apply the antibody via the recording pipette²⁹.

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